Methods for improved homologous recombination and compositions thereof

Abstract

The present disclosure relates to methods, kits, and compositions for improving the efficiency of homologous recombination. In particular, the disclosure relates to methods for cloning DNA molecules directly into a genome with the combined use of promoter trapping and short homology arms, nuclear localization signal, and/or binding one or more DNA binding agents (TAL effector domain or truncated guide RNA bound by Cas9) to specific sites thereby displacing or restructuring chromatin at the target locus, and/or it increasing the accessibility of the target locus to further enzymatic modifications. The methods and compositions provided herein are, inter alia, useful for genome editing and enhancing enzymatic processes involved therein.

1 . A method for altering an endogenous nucleic acid molecule present within a cell, the method comprising introducing a donor DNA molecule into the cell,
 wherein the donor DNA molecule is operably linked to one or more intracellular targeting moiety capable of localizing the donor DNA molecule to a location in the cell where the endogenous nucleic acid molecule is located.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the one or more intracellular target moiety is a nuclear localization signal. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the donor DNA molecule is single-stranded. 
     
     
         6 . The method of  claim 1 , wherein the donor DNA molecule has one or more nuclease resistant groups within 50 nucleotides of one or more terminus. 
     
     
         7 .- 10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the donor DNA molecule has two regions of sequence complementarity with a target locus present in the cell. 
     
     
         12 .- 19 . (canceled) 
     
     
         20 . A TALE protein comprising amino acids amino acids 811-830 of  FIG. 46 , wherein the amino acids at positions 815-816 and 824-825 may be Gly-Ser or Gly-Gly. 
     
     
         21 . The TALE protein of  claim 20  comprising amino acids amino acids 810-1029 of  FIG. 46 , wherein the amino acids at positions 1022-1023 may be Gly-Ser or Gly-Gly. 
     
     
         22 . The TALE protein of  claim 20  comprising amino acids amino acids 752-1021 of  FIG. 46 . 
     
     
         23 .- 28 . (canceled) 
     
     
         29 . A Cas9 protein comprising two or more bipartite nuclear localization signals. 
     
     
         30 .- 34 . (canceled) 
     
     
         35 . A method for engineering intracellular nucleic acid in a cell, the method comprising introducing into the cell the Cas9 protein of  claim 29  or nucleic acid encoding the Cas9 protein of  claim 29 , wherein the Cas9 protein is designed to bind to a target locus within the cell. 
     
     
         36 .- 37 . (canceled) 
     
     
         38 . A method for homologous recombination of an intracellular nucleic acid molecule at a cleavage site within a population of cells, the method comprising:
 (a) generating a double-stranded break in the intracellular nucleic acid molecule at the cleavage site to produce a cleaved nucleic acid molecule, and (b) contacting the cleaved nucleic acid molecule with a donor nucleic acid molecule,   wherein the donor nucleic acid molecule has at least ten nucleotides or base pairs of homology to nucleic acid located within 100 base pairs of each side of the cleavage site,   wherein at least 95% of the cells within the population of cells undergo homology directed repair with the donor nucleic acid molecule at the cleavage site.   
     
     
         39 .- 42  (canceled) 
     
     
         43 . The method of  claim 38 , wherein the donor DNA molecule is operably linked to one or more nuclear localization signals. 
     
     
         44 . The method of  claim 38 , wherein the population of cells is contacted with the one or more nucleic acid cutting entity or one of more nucleic acid molecule encoding one or more nucleic acid cutting entity, and the population of cells is subsequently contacted with one or more donor nucleic acid molecules 
     
     
         45 . The method of  claim 44 , wherein the population of cells is contacted with one or more donor nucleic acid molecules from  5  to  60  minutes after the population of cells is contacted with the one or more nucleic acid cutting entity or one of more nucleic acid molecules encoding one or more nucleic acid cutting entity. 
     
     
         46 . A method for homologous recombination in an initial nucleic acid molecule comprising:
 (a) generating a double-stranded break in the initial nucleic acid molecule to produce a cleaved nucleic acid molecule, and   (b) contacting the cleaved nucleic acid molecule with a donor nucleic acid molecules,
 wherein the initial nucleic acid molecule comprises a promoter and a gene, and 
   wherein the donor nucleic acid molecule comprises: (i) matched termini on the 5′ and 3′ ends of 12 bp to 250 bp in length, (ii) a promoterless selection marker, (iii) a reporter gene, (iv) either a self-cleaving peptide linking the promoterless selection marker and the reporter gene or a loxP on either side of the promoterless selection marker, and (iv) optionally a linker between the promoterless selection marker and the reporter gene.   
     
     
         47 . (canceled) 
     
     
         48 . The method of  claim 46 , wherein the double-stranded break is induced by one or more nucleic acid cutting entity. 
     
     
         49 .- 51 . (canceled) 
     
     
         52 . The method of  claim 46 , wherein the self-cleaving peptide is a self-cleaving 2A peptide. 
     
     
         53 .- 57 . (canceled) 
     
     
         58 . The method of  claim 46 , wherein the donor nucleic acid molecule is modified with one or more nuclease resistant groups in one or more strand of one or more terminus. 
     
     
         59 . (canceled) 
     
     
         60 . The method of  claim 46 , further comprising treating the donor nucleic acid molecule with one or more non-homologous end joining (NHEJ) inhibitor. 
     
     
         61 .- 62 . (canceled) 
     
     
         63 . A method of enhancing activity of a modulating protein or a modulating complex at a target locus in a cell, the method comprising:
 (1) introducing into a cell comprising a nucleic acid encoding the target locus:
 (i) a first modulating protein or a first modulating complex capable of binding a first modulator binding sequence of the target locus, wherein the first modulator binding sequence comprises a modulation site; and 
 (ii) a first DNA-binding modulation-enhancing agent capable of binding a first enhancer binding sequence of the target locus; and 
   (2) allowing the first DNA-binding modulation-enhancing agent to bind the first enhancer binding sequence, thereby enhancing activity of the first modulating protein or the first modulating complex at a target locus in a cell.   
     
     
         64 . The method of  claim 63 , wherein the introducing a first DNA-binding modulation-enhancing agent comprises introducing a vector encoding the first DNA-binding modulation-enhancing agent. 
     
     
         65 .- 66 . (canceled) 
     
     
         67 . The method of  claim 63 , further comprising:
 (1) introducing into the cell a second DNA-binding modulation-enhancing agent; and   (2) allowing the second DNA-binding modulation-enhancing agent to bind a second enhancer binding sequence of the target locus.   
     
     
         68 .- 69 . (canceled) 
     
     
         70 . The method of  claim 63 , wherein the first modulation protein is a DNA binding-nuclease fusion protein. 
     
     
         71 .- 78 . (canceled)