Improved 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 treating a genetic disorder in an organism, comprising
 introducing 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   into the organism.   
     
     
         2 . The method of  claim 1 , wherein the doNA molecule consists of DNA and the guide NA molecule consists of RNA, or wherein the fuNA consists of RNA. 
     
     
         3 . The method of  claim 1 , 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. 
     
     
         4 . 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. 
     
     
         5 . A method for treating a genetic disorder in an organism, comprising
 introducing a recombinant fusion nucleic acid (fuNA) molecule into the organism, wherein the fuNA molecule comprises a guide nucleic acid (gNA) molecule covalently linked to a donor nucleic acid (doNA) molecule into the organism,   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.   
     
     
         6 . The method of  claim 5 , 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. 
     
     
         7 . The method of  claim 6 , wherein wherein said scaffold NA forms a secondary structure comprising at least two hairpins. 
     
     
         8 . The method of  claim 1 , wherein the genetic disorder is sickle cell disease. 
     
     
         9 . The method of  claim 5 , wherein the genetic disorder is sickle cell disease.