Modified cascade ribonucleoproteins and uses thereof

Abstract

A clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for adaptive antiviral defence (Cascade); the Cascade protein complex comprising at least CRISPR-associated protein subunits Cas7, Cas5 and Cas6 which includes at least one subunit with an additional amino acid sequence possessing nucleic acid or chromatin modifying, visualising, transcription activating or transcription repressing activity. The Cascade complex with additional activity is combined with an RNA molecule to produce a ribonucleoprotein complex. The RNA molecule is selected to have substantial complementarity to a target sequence. Targeted ribonucleoproteins can be used as genetic engineering tools for precise cutting of nucleic acids in homologous recombination, non-homologous end joining, gene modification, gene integration, mutation repair or for their visualisation, transcriptional activation or repression. A pair of ribonucleotides fused to FokI dimers may be used to generate double-strand breakages in the DNA to facilitate these applications in a sequence-specific manner.

1 .- 46 . (canceled) 
     
     
         47 . A method for modifying a genomic target nucleic acid in a eukaryotic cell comprising:
 (a) contacting a nucleic acid-targeting nucleic acid with a target nucleic acid, wherein said nucleic acid-targeting nucleic acid comprises a designed clustered regularly interspaced short palindromic repeat RNA; and   (b) modifying said target nucleic acid in said eukaryotic cell.   
     
     
         48 . The method of  claim 47 , wherein said target nucleic acid is adjacent to a protospacer adjacent motif. 
     
     
         49 . The composition of  claim 48 , wherein said protospacer adjacent motif marks a point in said target nucleic acid at which base-pairing with said clustered regularly interspaced short palindromic repeat RNA occurs. 
     
     
         50 . The method of  claim 48 , wherein said protospacer adjacent motif comprises a triplet selected from the group consisting of: 5′-CCT-3′,5′-CTT-3′,5′-CAT-3′, and 5′-CTC-3′. 
     
     
         51 . The method of  claim 48 , wherein said protospacer adjacent motif comprises 5′-CCT-3′. 
     
     
         52 . The method of  claim 48 , wherein said protospacer adjacent motif comprises a CC dinucleotide. 
     
     
         53 . The method of  claim 48 , wherein said protospacer adjacent motif comprises a double-stranded triplet of nucleotides, wherein one strand said protospacer adjacent motif comprises a CC dinucleotide, wherein said CC dinucleotide is hybridized with a GG dinucleotide on the other strand of said protospacer adjacent motif. 
     
     
         54 . The method of  claim 47 , wherein said target nucleic acid is single stranded DNA. 
     
     
         55 . The method of  claim 47 , wherein said target nucleic acid is double stranded DNA. 
     
     
         56 . The method of  claim 47 , wherein said target nucleic acid is genomic DNA. 
     
     
         57 . The method of  claim 47 , wherein said nucleic acid-targeting nucleic acid is introduced into said eukaryotic cell by a delivery vehicle selected from the group consisting of: a cationic polymer and lipid delivery vehicle, a cell-penetrating peptide, a virus, an adenovirus, and an  agrobacterium.    
     
     
         58 . The method of  claim 47 , wherein said contacting comprises hybridizing a designed spacer portion of said clustered regularly interspaced short palindromic repeat RNA to a sequence of said target nucleic acid, wherein said designed spacer portion comprises at least 80% identity to said target nucleic acid. 
     
     
         59 . The method of  claim 47 , wherein said contacting comprises hybridizing a designed spacer portion of said clustered regularly interspaced short palindromic repeat RNA to a sequence of said target nucleic acid, wherein said designed spacer portion comprises at least 50% identity over 32 nucleotides of said target nucleic acid. 
     
     
         60 . The method of  claim 47 , wherein said nucleic acid-targeting nucleic acid comprises a seed region, a 3′ hairpin region, or both a seed region and a 3′ hairpin region. 
     
     
         61 . The method of  claim 47 , wherein said nucleic acid-targeting nucleic acid comprises a spacer region. 
     
     
         62 . The method of  claim 47 , wherein said modifying comprises introducing a single-stranded break in said target nucleic acid. 
     
     
         63 . The method of  claim 47 , wherein said modifying comprises introducing a double-stranded break in said target nucleic acid. 
     
     
         64 . The method of  claim 47 , wherein said modifying comprises introducing a modification selected from the group consisting of: an organic dye, a radiolabel, and a spin label. 
     
     
         65 . The method of  claim 47 , wherein said modifying comprises deleting a portion of said target nucleic acid. 
     
     
         66 . The method of  claim 47 , wherein said modifying is selected from the group consisting of: activating transcription of said target nucleic acid, repressing transcription of said target nucleic acid. 
     
     
         67 . The method of  claim 47 , wherein said modifying comprises inserting a nucleic acid into said target nucleic acid. 
     
     
         68 . The method of  claim 47 , wherein said modifying is performed by a Cas polypeptide. 
     
     
         69 . The method of  claim 68 , wherein said Cas polypeptide is a nuclease. 
     
     
         70 . The method of  claim 47 , wherein said cell is a cell selected from the group consisting of: an insect cell, a yeast cell, and a fungal cell. 
     
     
         71 . The method of  claim 47 , wherein said cell is a cell of a multicellular organism. 
     
     
         72 . The method of  claim 47 , wherein said cell is a mammalian cell. 
     
     
         73 . The method of  claim 47 , wherein said cell is a stem cell that is not a human embryonic stem cell. 
     
     
         74 . The method of  claim 47 , wherein said cell is a human cell. 
     
     
         75 . The method of  claim 47 , wherein said cell is a plant cell. 
     
     
         76 . A method of altering expression of a target nucleic acid comprising introducing into a eukaryotic cell containing a DNA molecule encoding said target nucleic acid a designed clustered regularly interspaced short palindromic repeat system comprising one or more vectors comprising: a) a first nucleotide sequence encoding a designed clustered regularly interspaced short palindromic repeat RNA that hybridizes with the target nucleic acid, and b) a second nucleotide sequence encoding a clustered regularly interspaced short palindromic repeat associated polypeptide, wherein components (a) and (b) are located on same or different vectors of the system, whereby said RNA targets said target nucleic acid and said clustered regularly interspaced short palindromic repeat associated polypeptide protein cleaves said DNA molecule.