Methods and means for genetic alteration of genomes utilizing designer dna recombining enzymes

Abstract

The invention provides methods and means for specifically altering the DNA sequence in a genome, in particular for genome editing by deleting or replacing a sequence of interest. Advantageously, the invention uses two non-identical sequences naturally occurring in a genome as target sites two which DNA-recombining enzymes are generated. The invention is in particular useful for medicine, in particular to repair a mutation in a genome or to delete predefined genetic material from cells or tissue and to cure diseases. An advantage of the invention is that it allows precise site directed altering of DNA without engaging host DNA repair pathways and thereby works without inducing random insertions and deletions (in-dels).

1 - 26 . (canceled) 
     
     
         27 . A method for site-specific DNA recombination, comprising introducing into a host cell a designer DNA-recombining enzyme, wherein the designer DNA-recombining enzyme comprises at least two different monomers, wherein the at least two different monomers recognize non-identical naturally occurring half sites in a genome of a host cell. 
     
     
         28 . The method of  claim 27 , wherein the recombination causes a deletion, an insertion, an inversion, or a replacement of genetic content. 
     
     
         29 . The method of  claim 27 , wherein the genome is a human genome. 
     
     
         30 . The method of  claim 27 , wherein the enzyme is capable of inducing a site-specific DNA recombination in a genome of a host cell by recombining two target sequences naturally occurring in the genome. 
     
     
         31 . The method of  claim 27 , wherein the target sequences are not identical or wherein the target sequences are identical. 
     
     
         32 . The method of  claim 27 , wherein the designer DNA-recombining enzyme is capable of causing a deletion, an insertion, an inversion, or a replacement of a nucleotide sequence in the genome of the host cell. 
     
     
         33 . The method of  claim 27 , wherein each target sequence comprises two half sites with an intervening spacer sequence. 
     
     
         34 . The method of  claim 27 , wherein the half sites are between 10-20 nucleotides in length. 
     
     
         35 . The method of  claim 27 , wherein the half sites are 13 nucleotides in length. 
     
     
         36 . The method of  claim 27 , wherein the half sites differ from each other by not more than 7 nucleotides. 
     
     
         37 . The method of  claim 27 , wherein the half sites differ from each other by not more than 5 nucleotides. 
     
     
         38 . The method of  claim 27 , wherein the half sites differ from each other by not more than 4 nucleotides. 
     
     
         39 . The method of  claim 27 , wherein the half sites differ in at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least eight nucleotides. 
     
     
         40 . The method of  claim 33 , wherein the spacer sequence is between 5 and 12 nucleotides in length. 
     
     
         41 . The method of  claim 33 , wherein the spacer sequence is 8 nucleotides in length. 
     
     
         42 . The method of  claim 33 , wherein the spacer sequence comprises a non-palindromic sequence having at least 2 positions of asymmetry. 
     
     
         43 . The method of  claim 33 , wherein the spacer sequence comprises a non-palindromic sequence having at least 4 positions of asymmetry. 
     
     
         44 . The method of  claim 33 , wherein the spacer sequence comprises a non-palindromic sequence having at least 6 positions of asymmetry. 
     
     
         45 . The method of  claim 33 , wherein each target sequence comprises an identical spacer sequence. 
     
     
         46 . The method of  claim 33 , wherein each target sequence comprises a non-identical spacer sequence.