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.

1 . An engineered CRISPR-Cas9 complex comprising a tracr mate sequence and a tracr sequence,
 wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two when aligned, wherein the complementary portions of the tracr and tracr mate sequences hybridize and have a length comprising about 16 to about 28 nucleotides, and the complex is designed to target a DNA sequence.   
     
     
         2 . The complex of  claim 1  wherein the tracr mate has a length of 28 nucleotides. 
     
     
         3 . The complex of  claim 1  wherein the tracr mate has a length of 16 nucleotides. 
     
     
         4 . Nucleic acid molecule(s) encoding the tracr mate and tracr of  claim 1 . 
     
     
         5 . Nucleic acid molecule(s) encoding the tracr mate and tracr of  claim 2 . 
     
     
         6 . Nucleic acid molecule(s) encoding the tracr mate and tracr of  claim 3 . 
     
     
         7 . A method of targeting a DNA molecule having a target sequence comprising:
 contacting the DNA molecule with an engineered CRISPR-Cas9 complex of  claim 1 .   
     
     
         8 . The method of  claim 7  wherein the tracr mate has a length of 28 nucleotides. 
     
     
         9 . The method of  claim 7  wherein the tracr mate has a length of 16 nucleotides. 
     
     
         10 . The complex of  claim 1 , which is not in a bacterial or archael cell. 
     
     
         11 . 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.   
     
     
         12 . The method of  claim 11 , wherein said modification of the target DNA molecule is cleavage of the target DNA molecule. 
     
     
         13 . The method of  claim 11 , wherein the target sequence is 15 nucleotides (nt) to 18 nt long. 
     
     
         14 . The method of  claim 11 , wherein the target sequence is 18 nucleotides (nt) to 25 nt long. 
     
     
         15 . The method of  claim 11 , wherein the target DNA molecule is chromosomal DNA. 
     
     
         16 . The method of  claim 11 , 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. 
     
     
         17 . The method of  claim 11 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of: (i) a non-natural intemucleoside 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. 
     
     
         18 . The method of  claim 11 , 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). 
     
     
         19 . The method of  claim 11 , 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. 
     
     
         20 . The method of  claim 11 , 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.