Methods for modification of target nucleic acids

Abstract

Methods for modification of target nucleic acids. The method involves a construct in which guide RNA is covalently linked to donor RNA (fusion NA) to be introduced into the target nucleic acid by homologous recombination and is based on the introduction of a nuclease, e.g. CRISPR or TALEN, into the cell containing the target nucleic acid. The fusion NA may be introduced as a DNA vector.

1 . A method for modification of a target nucleic acid (target NA) molecule in a cell comprising the steps of:
 a. providing a recombinant fusion nucleic acid (fuNA) molecule comprising a guide nucleic acid (gNA) molecule covalently linked to at least one donor nucleic acid (doNA) molecule, and   b. introducing said fuNA molecule into one or more cell(s) comprising the target NA molecule, and   c. introducing a site directed nucleic acid modifying polypeptide into said one or more cell(s), and   d. incubating the one or more cells under conditions that allow for homologous recombination in said one or more cell(s),   wherein the doNA comprises two homology arms and wherein said two homology arms are directly adjacent to each other.   
     
     
         2 . The method of  claim 1 , further comprising isolating one or more cells in which homologous recombination occurred. 
     
     
         3 . The method of  claim 1 , wherein, the doNA molecule and gNA molecule consist of RNA, DNA, or PNA. 
     
     
         4 . The method of  claim 1 , wherein the doNA molecule consists of DNA and the gNA molecule consists of RNA, or wherein the fuNA consists of RNA. 
     
     
         5 . The method of  claim 1 , wherein each homology arm independently comprises at least 15 bases complementary to a different area of at least 15 consecutive bases of the target NA molecule from the other homology arm, wherein the homology arms each comprise at least 15 bases that are 100% complementary to the same number of consecutive bases in the target NA molecule. 
     
     
         6 . The method of  claim 1 , wherein one or both of the homology arms comprise more than 15 bases, and wherein the homology arm(s) comprising more than 15 bases is/are at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementary to an area of the target NA molecule. 
     
     
         7 . The method of  claim 1 , wherein the gNA comprises a spacer NA and a scaffold NA molecule, wherein the scaffold NA forms a secondary structure comprising at least one hairpin, and wherein the spacer NA comprises at least 18 bases 100% complementary to the same number of consecutive bases of the target NA molecule. 
     
     
         8 . The method of  claim 1 , wherein the cell is a microbial, animal, human or plant cell. 
     
     
         9 . The method of  claim 1 , wherein the site directed nucleic acid modifying polypeptide has a double-stranded nucleic acid digestion function, or a nickase function, cutting only one strand of a double-stranded nucleic acid molecule. 
     
     
         10 . The method of  claim 1 , wherein the fuNA molecule is a fuRNA molecule and is introduced into the cell or composition comprising the target NA molecule as an RNA molecule or as one or more expression constructs encoding said fuRNA molecule. 
     
     
         11 . A recombinant fuNA molecule comprising a doNA molecule covalently linked to a gNA molecule, wherein the doNA comprises two homology arms each independently comprising at least 15 bases complementary to a different area of at least 15 consecutive bases of the target NA molecule from the other homology arm, and wherein said two homology arms are directly adjacent to each other. 
     
     
         12 . The recombinant fuNA molecule of  claim 11 , wherein the doNA molecule consists of DNA and the guide NA molecule consists of RNA, or wherein the fuNA consists of RNA. 
     
     
         13 . The recombinant fuNA molecule of  claim 11 , wherein the homology arms each comprise at least 15 bases are 100% complementary to the same number of consecutive bases in the target NA molecule, and wherein, in the event a homology arm is larger than 15 bases, it is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% complementary to the target NA molecule. 
     
     
         14 . The recombinant fuNA molecule of  claim 11 , wherein the gNA comprises a spacer NA and a scaffold NA molecule, wherein the scaffold NA forms a secondary structure comprising at least one hairpin, and wherein the spacer NA comprises at least 18 bases 100% complementary to the same number of consecutive bases of the target NA molecule. 
     
     
         15 . A recombinant fusion nucleic acid (fuNA) molecule comprising a guide nucleic acid (gNA) molecule covalently linked to a donor nucleic acid (doNA) molecule,
 wherein said fuNA molecule consists of RNA,   wherein said doNA molecule comprises two homology arms,   wherein each of said two homology arms independently comprises at least 15 bases complementary to a different area of at least 15 consecutive bases of the target NA molecule from the other homology arm,   wherein at least 15 bases are 100% complementary to the same number of consecutive bases in the target NA molecule,   wherein both homology arms are separated by at least one or more bases or wherein both homology arms are directly adjacent to each other, and   wherein both homology arms have the same length or different lengths.   
     
     
         16 . The recombinant fuNA molecule of  claim 15 , wherein the gNA molecule comprises a spacer nucleic acid (spacer NA) molecule and a scaffold nucleic acid (scaffold NA) molecule, wherein said spacer NA molecule comprises at least 12 bases, at least 13 bases, at least 14 bases, at least 15 bases, at least 16 bases, at least 17 bases, at least 18 bases, at least 19 bases, or at least 20 bases which are 100% complementary to the target NA molecule, and wherein said scaffold NA forms a secondary structure comprising at least one hairpin, particularly preferably at least two hairpins. 
     
     
         17 . A vector comprising an expression construct comprising a promoter functionally linked to a DNA molecule encoding the fuNA molecule of  claim 11 . 
     
     
         18 . A vector system comprising the vector of  claim 17  and a vector encoding a site directed nucleic acid modifying polypeptide. 
     
     
         19 . A system for modification of a target NA molecule in a cell comprising
 a. the vector of  claim 17 ,   b. a vector encoding a site directed nucleic acid modifying polypeptide, and   c. a cell comprising a target NA molecule.   
     
     
         20 . A composition comprising
 a. the vector of  claim 17 ,   b. a vector encoding a site directed nucleic acid modifying polypeptide, and   c. a cell comprising a target NA molecule.