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 the treatment of a genetic disease comprising administering a composition comprising:
 a. 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. a component selected from one or more of:
 i. a nucleic acid molecule encoding a site directed nucleic acid modifying polypeptide, or 
 ii. a site directed nucleic acid modifying polypeptide. to an organism in need thereof. 
   
     
     
         2 . The method of  claim 1 , wherein wherein the gNA comprises a spacer nucleic acid (spacer NA) molecule, wherein said spacer NA molecule comprises at least 12 bases which are 100% complementary to the same number of consecutive bases of the target NA molecule. 
     
     
         3 . The method of  claim 2 , wherein said spacer NA molecule comprises 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 complementary to the target NA molecule. 
     
     
         4 . The method of  claim 1 , wherein said doNA molecule and gNA molecule consist of RNA, DNA, or PNA. 
     
     
         5 . 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. 
     
     
         6 . The method of  claim 1 , wherein the at least one 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, and wherein both homology arms have the same length or different lengths. 
     
     
         7 . The method of  claim 1 , wherein the two homology arms are separated by at least one or more bases, or wherein both homology arms are directly adjacent to each other. 
     
     
         8 . The method of  claim 1 , wherein the gNA molecule further comprises a scaffold nucleic acid (scaffold NA) molecule, wherein said scaffold NA forms a secondary structure comprising at least one hairpin. 
     
     
         9 . The method of  claim 1 , wherein the gNA comprises a scaffold NA and wherein said scaffold NA molecule is covalently bound to the gNA molecule. 
     
     
         10 . The method of  claim 1 , wherein the site directed nucleic acid modifying polypeptide is a nucleic acid guided nucleic acid modifying polypeptide or a functional fragment thereof. 
     
     
         11 . The method of  claim 1 , wherein said site directed nucleic acid modifying polypeptide has a nickase function or is an inactivated site directed nucleic acid modifying polypeptide or an inactivated site directed nucleic acid modifying polypeptide linked to other functional groups. 
     
     
         12 . The method of  claim 1 , further comprising a cell comprising a target NA molecule, wherein said cell is a microbial, animal, human or plant cell. 
     
     
         13 . The method of  claim 1 , wherein the composition consists of
 (i) the recombinant fusion nucleic acid (fuNA) molecule according to  claim 1 , wherein the gNA molecule comprises a spacer nucleic acid (spacer NA) molecule, which 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, at least 19 bases, or at least 20 bases which are 100% complementary to the same number of consecutive bases of the target NA molecule,   and   (ii) a component selected from one or more of
 (a) a nucleic acid molecule encoding a site directed nucleic acid modifying polypeptide, wherein said site directed nucleic acid modifying polypeptide has a nickase function or is an inactivated site directed nucleic acid modifying polypeptide or an inactivated site directed nucleic acid modifying polypeptide linked to other functional groups and wherein said nucleic acid molecule consists of RNA 
 Or 
 (b) a site directed nucleic acid modifying polypeptide, wherein said site directed nucleic acid modifying polypeptide has a nickase function or is an inactivated site directed nucleic acid modifying polypeptide or an inactivated site directed nucleic acid modifying polypeptide linked to other functional groups and 
 and 
   (iii) a cell comprising a target nucleic acid (target NA) molecule.   
     
     
         14 . The method of  claim 13 , wherein said site directed nucleic acid modifying polypeptide is a nucleic acid guided nucleic acid modifying polypeptide or a functional fragment thereof. 
     
     
         15 . The method of  claim 13 , wherein the cell is a microbial, animal, human or plant cell. 
     
     
         16 . The method of  claim 8 , wherein said scaffold NA forms a secondary structure comprising at least two hairpins. 
     
     
         17 . The method of  claim 1 , wherein the disease is selected from one or more of:
 (i) viral infections,   (ii) hematologic disorders,   (iii) neuromuscular disorders,   (iv) skin disorders,   (v) ocular disorders, or   (vi) respiratory disorders.   
     
     
         18 . The method of  claim 17 , wherein the disease is selected from one or more of:
 (i) wherein the viral infection is HIV, Hepatitis B virus, Herpes simplex virus, and/or Human papilloma virus,   (ii) wherein the hematologic disorder is X-SCID, ADA-SCID, RS-SCID, sickle cell disease, and/or/β-thalessemia,   (iii) wherein the neuromuscular disorder is Duchenne muscular dystrophy,   (iv) wherein the skin disorder is epidermolysis bullosa,   (v) wherein the ocular disorder is Leber's Congenital Amaurosis type 10, and/or   (vi) wherein the respiratory disorder is cystic fibrosis.   
     
     
         19 . The method of  claim 17 , wherein the doNA molecule comprises two homology arms and wherein both homology arms are directly adjacent to each other.