Crispr-cas component systems, methods and compositions for sequence manipulation

Abstract

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.

What is claimed is: 
     
         1 . A method of modifying a target DNA molecule, the method comprising:
 contacting a target DNA molecule having a target sequence with a complex comprising:   (a) a Cas9 protein; and   (b) a DNA-targeting RNA comprising:
 (i) a targeter-RNA that hybridizes with the target sequence, and 
 (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, 
   wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 10 to 15 base pairs,   wherein said contacting takes place outside of a bacterial cell and outside of an archaeal cell, thereby resulting in modification of the target DNA molecule.   
     
     
         2 . The method of  claim 1 , wherein said modification of the target DNA molecule is cleavage of the target DNA molecule. 
     
     
         3 . The method of  claim 1 , wherein the target sequence is 15 nucleotides (nt) to 18 nt long. 
     
     
         4 . The method of  claim 1 , wherein the target sequence is 18 nucleotides (nt) to 25 nt long. 
     
     
         5 . The method of  claim 1 , wherein the target DNA molecule is chromosomal DNA. 
     
     
         6 . The method of  claim 1 , wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence set forth in SEQ ID NO: 441. 
     
     
         7 . The method of  claim 1 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase. 
     
     
         8 . The method of  claim 1 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of: (i) a non-natural internucleoside linkage selected from a phosphorothioate, an inverted polarity linkage, and an abasic nucleoside linkage; (ii) a locked nucleic acid (LNA); and (iii) a modified sugar moiety selected from 2′-O-methoxyethyl, 2′-O-methyl, and 2′-fluoro. 
     
     
         9 . The method of  claim 1 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of: a peptide nucleic acid (PNA), a morpholino nucleic acid, a cyclohexenyl nucleic acid (CeNA), and/or a locked nucleic acid (LNA). 
     
     
         10 . The method of  claim 1 , wherein the targeter-RNA and/or the activator-RNA comprises one or more modified sugar moieties selected from: 2′-O-(2-methoxyethyl), 2′-dimethylaminooxyethoxy, 2′-dimethylaminoethoxyethoxy, 2′-O-methyl, and 2′-fluoro. 
     
     
         11 . The method of  claim 1 , wherein the targeter-RNA and/or the activator-RNA is conjugated to a moiety selected from: a polyamine; a polyamide; a polyethylene glycol; a polyether; a cholesterol moiety; a cholic acid; a thioether; a thiocholesterol; an aliphatic chain; a phospholipid; an adamantane acetic acid; a palmityl moiety; an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety; a biotin; a phenazine; a folate; a phenanthridine; an anthraquinone; an acridine; a fluorescein; a rhodamine; a fluor; and a coumarin.