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 Fold 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 composition comprising:
 (a) a double-stranded target nucleic acid comprising a protospacer adjacent motif; and   (b) a synthetically constructed non-natural nucleic acid comprising a CRISPR RNA, wherein a spacer portion of said CRISPR RNA is hybridized with one strand of said double-stranded target nucleic acid.   
     
     
         48 . The composition of  claim 47 , wherein said synthetically constructed non-natural nucleic acid comprising a CRISPR RNA is a designed non-natural nucleic acid comprising a CRISPR RNA. 
     
     
         49 . The composition of  claim 47 , wherein said spacer portion of said CRISPR RNA is hybridized adjacent to said protospacer adjacent motif. 
     
     
         50 . The composition of  claim 47 , further comprising a CRISPR associated polypeptide. 
     
     
         51 . The composition of  claim 50 , wherein said CRISPR associated polypeptide is a nuclease. 
     
     
         52 . The composition of  claim 50 , wherein said CRISPR associated polypeptide forms a complex with said double-stranded target nucleic acid. 
     
     
         53 . The composition of  claim 50 , wherein said CRISPR associated polypeptide forms a complex with said CRISPR RNA. 
     
     
         54 . The composition of  claim 47 , wherein said protospacer adjacent motif is located at the 5′ end of said double-stranded target nucleic acid. 
     
     
         55 . The composition of  claim 47 , wherein said protospacer adjacent motif is located at the 3′ end of said double-stranded target nucleic acid. 
     
     
         56 . The composition of  claim 47 , wherein said protospacer adjacent motif comprises 5′-CTT-3′. 
     
     
         57 . The composition of  claim 47 , wherein said protospacer adjacent motif comprises 5′-CAT-3′. 
     
     
         58 . The composition of  claim 47 , wherein said protospacer adjacent motif comprises 5′-CTC-3′. 
     
     
         59 . The composition of  claim 47 , wherein said protospacer adjacent motif comprises 5′-CCT-3′. 
     
     
         60 . The composition of  claim 47 , wherein one strand of said protospacer adjacent motif comprises a CC dinucleotide. 
     
     
         61 . The composition of  claim 47 , 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. 
     
     
         62 . The composition of  claim 47 , wherein said double-stranded target nucleic acid is DNA. 
     
     
         63 . The composition of  claim 47 , wherein said double-stranded target nucleic acid is a target for genetic modification. 
     
     
         64 . The composition of  claim 47 , wherein said spacer portion of said CRISPR RNA hybridizes to said double-stranded target nucleic acid with a dissociation constant from 1 picomolar to 1 micromolar. 
     
     
         65 . The composition of  claim 47 , wherein said spacer portion of said CRISPR RNA hybridizes to said double-stranded target nucleic acid with a dissociation constant from 1 nanomolar to 100 nanomolar. 
     
     
         66 . The composition of  claim 47 , wherein said spacer portion of said CRISPR RNA is synthetically constructed to hybridize to a sequence of said double-stranded target nucleic acid comprising at least 50% identity to said spacer portion. 
     
     
         67 . The composition of  claim 47 , wherein said CRISPR RNA comprises a handle region. 
     
     
         68 . The composition of  claim 47 , wherein said CRISPR RNA comprises a seed region. 
     
     
         69 . The composition of  claim 47 , wherein said CRISPR RNA comprises a 3′ hairpin region. 
     
     
         70 . The composition of  claim 47 , wherein said CRISPR RNA comprises a handle region, a seed region, and 3′ hairpin region, or any combination thereof. 
     
     
         71 . The composition of  claim 70 , wherein said handle region is located 5′ to said seed region which is located 5′ to said spacer portion which is located 5′ to said 3′ hairpin region. 
     
     
         72 . The composition of  claim 50 , wherein said CRISPR associated polypeptide is adapted to induce a modification in said double-stranded target nucleic acid. 
     
     
         73 . The composition of  claim 50 , wherein said CRISPR associated polypeptide further comprises a polypeptide selected from the group consisting of: helicase, nuclease, methyltransferase, demethylase, acetylase, deacetylase, phosphatase, kinase, transcription activator, RNA polymerase subunit, transcription repressor, DNA binding polypeptide, DNA structuring polypeptide, marker polypeptide, reporter polypeptide, fluorescent polypeptide, ligand binding polypeptide, signal polypeptide, subcellular localization polypeptide, and antibody epitope, or any combination thereof. 
     
     
         74 . The composition of  claim 50 , wherein said CRISPR associated polypeptide further comprises a nuclease. 
     
     
         75 . The composition of  claim 50 , wherein said CRISPR associated polypeptide further comprises a helicase. 
     
     
         76 . The composition of  claim 50 , wherein said CRISPR associated polypeptide further comprises a transcription activator. 
     
     
         77 . The composition of  claim 50 , wherein said CRISPR associated polypeptide further comprises a transcription repressor. 
     
     
         78 . The composition of  claim 47 , wherein said double-stranded target nucleic acid is bound to a solid support. 
     
     
         79 . A method of synthesizing a synthetically constructed non-natural CRISPR RNA comprising: synthesizing the synthetically constructed non-natural nucleic acid of  claim 47 . 
     
     
         80 . A composition comprising: a nucleic acid-targeting nucleic acid comprising:
 (a) a synthetically constructed non-natural spacer region adapted to hybridize to one strand of a double-stranded target nucleic acid comprising a protospacer adjacent motif;   (b) a nucleic acid duplex;   (c) a linker linking both strands of said duplex,   wherein at least one strand of said duplex is adapted to form a complex with a CRISPR associated polypeptide.   
     
     
         81 . The composition of  claim 80 , wherein said duplex comprises a hairpin. 
     
     
         82 . The composition of  claim 81 , wherein said duplex is 3′ to said synthetically constructed non-natural spacer region. 
     
     
         83 . The composition of  claim 80 , wherein said linker comprises a tetranucleotide loop. 
     
     
         84 . The composition of  claim 80 , wherein said synthetically constructed non-natural spacer region is 5′ to said duplex. 
     
     
         85 . The composition of  claim 80 , wherein said synthetically constructed non-natural spacer region hybridizes adjacent to said protospacer adajcent motif of said one strand of said double-stranded target nucleic acid. 
     
     
         86 . The composition of  claim 80 , wherein said spacer region comprises a seed region. 
     
     
         87 . The composition of  claim 80 , wherein said CRISPR associated polypeptide is a nuclease. 
     
     
         88 . The composition of  claim 80 , wherein said protospacer adjacent motif is located at the 5′ end of said double-stranded target nucleic acid. 
     
     
         89 . The composition of  claim 80 , wherein said protospacer adjacent motif is located at the 3′ end of said double-stranded target nucleic acid. 
     
     
         90 . The composition of  claim 80 , wherein said protospacer adjacent motif comprises 5′-CTT-3′. 
     
     
         91 . The composition of  claim 80 , wherein said protospacer adjacent motif comprises 5′-CAT-3′. 
     
     
         92 . The composition of  claim 80 , wherein said protospacer adjacent motif comprises 5′-CTC-3′. 
     
     
         93 . The composition of  claim 80 , wherein said protospacer adjacent motif comprises 5′-CCT-3′. 
     
     
         94 . The composition of  claim 80 , wherein one strand of said protospacer adjacent motif comprises a CC dinucleotide. 
     
     
         95 . The composition of  claim 80 , 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. 
     
     
         96 . The composition of  claim 80 , wherein said double-stranded target nucleic acid is DNA. 
     
     
         97 . The composition of  claim 80 , wherein said double-stranded target nucleic acid is a target for genetic modification. 
     
     
         98 . The composition of  claim 80 , wherein said synthetically constructed spacer region hybridizes to said double-stranded target nucleic acid with a dissociation constant from 1 picomolar to 1 micromolar. 
     
     
         99 . The composition of  claim 80 , wherein said synthetically constructed spacer region hybridizes to said double-stranded target nucleic acid with a dissociation constant from 1 nanomolar to 100 nanomolar. 
     
     
         100 . The composition of  claim 80 , wherein said synthetically constructed spacer region is synthetically constructed to hybridize to a sequence of said double-stranded target nucleic acid comprising at least 50% identity to said synthetically constructed spacer region. 
     
     
         101 . The composition of  claim 80 , wherein said nucleic acid-targeting nucleic acid is from 35-75 nucleotides in length. 
     
     
         102 . The composition of  claim 80 , further comprising a delivery vehicle. 
     
     
         103 . The composition of  claim 102 , wherein said delivery vehicle comprises a virus. 
     
     
         104 . The composition of  claim 102 , wherein said delivery vehicle comprises adenovirus. 
     
     
         105 . The composition of  claim 102 , wherein said delivery vehicle comprises an  Agrobacterium.    
     
     
         106 . A pharmaceutical composition comprising the composition of  claim 80 . 
     
     
         107 . A method of synthesizing a synthetically constructed non-natural CRISPR RNA comprising: synthesizing the synthetically constructed non-natural nucleic acid of  claim 80 . 
     
     
         108 . The composition of  claim 80  for use as a medicament. 
     
     
         109 . A composition comprising: a delivery vehicle comprising a CRISPR RNA, wherein a portion of said CRISPR RNA is adapted to hybridize to a region of one strand of a double-stranded target nucleic acid, wherein said region is adjacent to a protospacer adjacent motif. 
     
     
         110 . The composition of  claim 109 , wherein said delivery vehicle is selected from the group consisting of: cationic polymers, lipids, and cell-penetrating peptide, or any combination thereof. 
     
     
         111 . The composition of  claim 109 , wherein said delivery vehicle comprises a virus. 
     
     
         112 . The composition of  claim 109 , wherein said delivery vehicle comprises adenovirus. 
     
     
         113 . The composition of  claim 109 , wherein said delivery vehicle comprises an  Agrobacterium.    
     
     
         114 . A pharmaceutical composition comprising the composition of  claim 109 . 
     
     
         115 . The composition of  claim 109  for use as a medicament. 
     
     
         116 . A vector comprising:
 (a) a first polynucleotide sequence encoding a CRISPR associated polypeptide; and   (b) a second polynucleotide sequence encoding a visualizing polypeptide.   
     
     
         117 . The vector of  claim 116 , wherein said CRISPR associated polypeptide comprises nuclease activity. 
     
     
         118 . The vector of  claim 116 , wherein said vector comprises a third polynucleotide sequence encoding for a linker, wherein said third polynucleotide sequence is between said first and second polynucleotide sequences. 
     
     
         119 . The vector of  claim 116 , wherein said vector further comprises a polynucleotide sequence encoding for a CRISPR RNA. 
     
     
         120 . The vector of  claim 116 , wherein said visualizing polypeptide comprises a fluorescent protein. 
     
     
         121 . The vector of  claim 116 , wherein said visualizing polypeptide comprises green fluorescent protein. 
     
     
         122 . The vector of  claim 116 , wherein said visualizing polypeptide comprises yellow fluorescent protein. 
     
     
         123 . A pharmaceutical composition comprising: the vector of  claim 116 . 
     
     
         124 . A delivery vehicle comprising: the vector of  claim 116 . 
     
     
         125 . The delivery vehicle of  claim 124 , wherein said delivery vehicle is selected from the group consisting of: cationic polymers, lipids, and cell-penetrating peptide, or any combination thereof. 
     
     
         126 . The delivery vehicle of  claim 124 , wherein said delivery vehicle comprises a virus. 
     
     
         127 . The delivery vehicle of  claim 124 , wherein said delivery vehicle comprises adenovirus. 
     
     
         128 . The delivery vehicle of  claim 124 , wherein said delivery vehicle comprises an  Agrobacterium.    
     
     
         129 . 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 CRISPR RNA; and   (b) modifying said target nucleic acid in said eukaryotic cell.   
     
     
         130 . The method of  claim 129 , wherein said target nucleic acid is adjacent to a protospacer adjacent motif. 
     
     
         131 . The method of  claim 129 , wherein a spacer of said nucleic acid-targeting nucleic acid is synthetically constructed. 
     
     
         132 . The method of  claim 129 , wherein said target nucleic acid is single stranded DNA. 
     
     
         133 . The method of  claim 129 , wherein said target nucleic acid is double stranded DNA. 
     
     
         134 . The method of  claim 129 , wherein said target nucleic acid is RNA. 
     
     
         135 . The method of  claim 129 , wherein said target nucleic acid is genomic DNA. 
     
     
         136 . The method of  claim 129 , wherein said contacting comprises hybridizing a portion of said nucleic acid-targeting nucleic acid to said target nucleic acid. 
     
     
         137 . The method of  claim 129 , wherein said contacting comprises binding said nucleic acid-targeting nucleic acid to said target nucleic acid with a dissociation constant of at least 1 micromolar. 
     
     
         138 . The method of  claim 129 , wherein said nucleic acid-targeting nucleic acid is introduced into said eukaryotic cell by a delivery method selected from the group consisting of: microinjection, transfection, electroporation, calcium co-precipitation, cationic polymer and lipid delivery, and cell-penetrating particles, or any combination thereof. 
     
     
         139 . The method of  claim 129 , wherein said contacting comprises hybridizing a synthetically constructed spacer portion of said CRISPR RNA to a sequence of said target nucleic acid comprising at least 50% identity to said synthetically constructed spacer portion. 
     
     
         140 . The method of  claim 129 , wherein said nucleic acid-targeting nucleic acid is RNA. 
     
     
         141 . The method of  claim 129 , wherein said nucleic acid-targeting nucleic acid comprises a handle region. 
     
     
         142 . The method of  claim 129 , wherein said nucleic acid-targeting nucleic acid comprises a seed region. 
     
     
         143 . The method of  claim 129 , wherein said nucleic acid-targeting nucleic acid comprises a spacer region. 
     
     
         144 . The method of  claim 129 , wherein said nucleic acid-targeting nucleic acid comprises a 3′ hairpin region. 
     
     
         145 . The method of  claim 129 , wherein said nucleic acid-targeting nucleic acid comprises a handle region, a seed region, a spacer region, and 3′ hairpin region, or any combination thereof. 
     
     
         146 . The method of  claim 145 , wherein said handle region is located 5′ to said seed region which is located 5′ to said spacer region which is located 5′ to said 3′ hairpin region. 
     
     
         147 . The method of  claim 129 , wherein said modifying comprises cleaving said target nucleic acid. 
     
     
         148 . The method of  claim 129 , wherein said modifying comprises introducing a single-stranded break in said target nucleic acid. 
     
     
         149 . The method of  claim 129 , wherein said modifying comprises introducing a double-stranded break in said target nucleic acid. 
     
     
         150 . The method of  claim 129 , wherein said modifying comprises allowing said target nucleic acid to be visualized. 
     
     
         151 . The method of  claim 129 , wherein said modifying comprises introducing a modification selected from the group consisting of: an organic dye, a radiolabel, and a spin label, or any combination thereof. 
     
     
         152 . The method of  claim 129 , wherein said modifying comprises deleting a portion of said target nucleic acid. 
     
     
         153 . The method of  claim 129 , wherein said modifying is selected from the group consisting of: activating trancription of said target nucleic acid, repressing transcription of said target nucleic acid, or both activating and repressing transcription of said target nucleic acid. 
     
     
         154 . The method of  claim 129 , wherein said modifying comprises inserting a nucleic acid into said target nucleic acid. 
     
     
         155 . The method of  claim 129 , wherein said modifying is performed by a CRISPR associated polypeptide. 
     
     
         156 . The method of  claim 155 , wherein said CRISPR associated polypeptide is a nuclease. 
     
     
         157 . The method of  claim 129 , wherein said cell is an insect cell. 
     
     
         158 . The method of  claim 129 , wherein said cell is a cell of a multicellular organism. 
     
     
         159 . The method of  claim 129 , wherein said cell is a mammalian cell. 
     
     
         160 . The method of  claim 129 , wherein said cell is a mammalian stem cell. 
     
     
         161 . The method of  claim 129 , wherein said cell is a human cell. 
     
     
         162 . The method of  claim 129 , wherein said cell is a human stem cell. 
     
     
         163 . The method of  claim 129 , wherein said cell is a plant cell. 
     
     
         164 . The method of  claim 129 , wherein said cell is an isolated cell. 
     
     
         165 . The method of  claim 164 , wherein said cell is a yeast cell. 
     
     
         166 . The method of  claim 164 , wherein said cell is a fungal cell. 
     
     
         167 . A composition comprising:
 (a) a eukaryotic cell;   (b) a double-stranded target nucleic acid in said eukaryotic cell comprising a protospacer adjacent motif; and   (c) a non-natural nucleic acid comprising a CRISPR RNA, wherein a spacer portion of said CRISPR RNA is hybridized with one strand of said double-stranded target nucleic acid in said eukaryotic cell.   
     
     
         168 . The eukaryotic cell of  claim 167 , wherein a portion of said CRISPR RNA hybridizes to one strand of a double-stranded target nucleic acid in said eukaryotic cell. 
     
     
         169 . The eukaryotic cell of  claim 167 , wherein said cell is an insect cell. 
     
     
         170 . The eukaryotic cell of  claim 167 , wherein said cell is a cell of a multicellular organism. 
     
     
         171 . The eukaryotic cell of  claim 167 , wherein said cell is a mammalian cell. 
     
     
         172 . The eukaryotic cell of  claim 167 , wherein said cell is a mammalian stem cell. 
     
     
         173 . The eukaryotic cell of  claim 167 , wherein said cell is a human cell. 
     
     
         174 . The eukaryotic cell of  claim 167 , wherein said cell is a human stem cell. 
     
     
         175 . The eukaryotic cell of  claim 167 , wherein said cell is a plant cell. 
     
     
         176 . The eukaryotic cell of  claim 167 , wherein said cell is a cell of a multicellular tissue. 
     
     
         177 . The eukaryotic cell of  claim 167 , wherein said cell is an isolated cell. 
     
     
         178 . The eukaryotic cell of  claim 167 , wherein said cell is a cell of an organ. 
     
     
         179 . The eukaryotic cell of  claim 167 , wherein said cell is a cell of an organism. 
     
     
         180 . A method for using a eukaryotic cell comprising:
 (a) isolating said eukaryotic cell;   (b) contacting a target nucleic acid in said cell with a complex comprising:
 (i) a CRISPR RNA; and 
 (ii) a CRISPR associated polypeptide; 
   (c) modifying said target nucleic acid; and   (d) relocating said eukaryotic cell.   
     
     
         181 . The method of  claim 180 , wherein said isolating comprises isolating from a whole tissue. 
     
     
         182 . The method of  claim 180 , wherein said isolating comprises isolating from an organ. 
     
     
         183 . The method of  claim 180 , wherein said isolating comprises isolating from an organism. 
     
     
         184 . The method of  claim 180 , wherein said relocating comprises transplanting said cell to a different location from which said cell originated. 
     
     
         185 . The method of  claim 180 , wherein said relocating comprises transplanting said cell to a different organism from which said cell originated. 
     
     
         186 . The method of  claim 180 , wherein said relocating comprises transplanting said cell to a same location from which said cell originated. 
     
     
         187 . The method of  claim 180 , wherein said relocating comprises transplanting said cell to a same organism from which said cell originated. 
     
     
         188 . The method of  claim 180 , wherein said relocating comprises a transplanting method selected from the group consisting of: allograft transplant, autograft transplant, isograft transplant, and xenograft transplant, or any combination thereof. 
     
     
         189 . The method of  claim 180 , wherein said contacting comprises hybridizing a portion of said nucleic acid-targeting nucleic acid to said target nucleic acid. 
     
     
         190 . The method of  claim 180 , wherein said contacting comprises hybridizing a portion of said CRISPR RNA to said target nucleic acid with a dissociation constant from 1 picomolar to 1 micromolar. 
     
     
         191 . The method of  claim 180 , wherein said contacting comprises hybridizing a portion of said CRISPR RNA to said target nucleic acid with a dissociation constant from 1 nanomolar to 100 nanomolar. 
     
     
         192 . The method of  claim 180 , wherein said contacting comprises introducing said nucleic acid-targeting nucleic acid by a delivery method selected from the group consisting of: microinjection, transfection, electroporation, calcium co-precipitation, cationic polymer and lipid delivery, and cell-penetrating particles, or any combination thereof. 
     
     
         193 . The method of  claim 180 , wherein said contacting comprises contacting said complex to said target nucleic acid at a site adjacent to a protospacer adjacent motif. 
     
     
         194 . The method of  claim 193 , wherein said protospacer adjacent motif is located 5′ to said target nucleic acid. 
     
     
         195 . The method of  claim 193 , wherein said protospacer adjacent motif is located 3′ to said target nucleic acid. 
     
     
         196 . The method of  claim 193 , wherein said protospacer adjacent motif comprises 5′-CTT-3′. 
     
     
         197 . The method of  claim 193 , wherein said protospacer adjacent motif comprises 5′-CAT-3′. 
     
     
         198 . The method of  claim 193 , wherein said protospacer adjacent motif comprises 5′-CTC-3′. 
     
     
         199 . The method of  claim 193 , wherein said protospacer adjacent motif comprises 5′-CCT-3′. 
     
     
         200 . The method of  claim 180 , wherein said target nucleic acid is selected from the group consisting of ssDNA, dsDNA, and RNA. 
     
     
         201 . The method of  claim 180 , wherein said target nucleic acid is DNA. 
     
     
         202 . The method of  claim 180 , wherein said CRISPR associated polypeptide is a nuclease. 
     
     
         203 . The method of  claim 180 , wherein said CRISPR associated polypeptide further comprises a polypeptide selected from the group consisting of: helicase, nuclease, methyltransferase, demthylase, acetylase, deacetylase, phosphatase, kinase, transcription activator, RNA polymerase subunit, transcription repressor, DNA binding polypeptide, DNA structuring polypeptide, marker polypeptide, reporter polypeptide, fluorescent polypeptide, ligand binding polypeptide, signal polypeptide, subcellular localization polypeptide, and antibody epitope, or any combination thereof. 
     
     
         204 . The method of  claim 180 , wherein said CRISPR RNA comprises a handle region. 
     
     
         205 . The method of  claim 180 , wherein said CRISPR RNA comprises a seed region. 
     
     
         206 . The method of  claim 180 , wherein said CRISPR RNA comprises a 3′ hairpin region. 
     
     
         207 . The method of  claim 180 , wherein said CRISPR RNA comprises a handle region, a seed region, a spacer region and 3′ hairpin region, or any combination thereof. 
     
     
         208 . The method of  claim 207 , wherein said handle region is located 5′ to said seed region which is located 5′ to said spacer region, which is located 5′ to said 3′ hairpin region. 
     
     
         209 . The method of  claim 180 , wherein said contacting comprises hybridizing a synthetically constructed spacer portion of said CRISPR RNA to a sequence of said target nucleic acid comprising at least 50% identity to said synthetically constructed spacer portion. 
     
     
         210 . The method of  claim 180 , wherein said nucleic acid-targeting nucleic acid is RNA. 
     
     
         211 . The method of  claim 180 , wherein said modifying comprises cleaving said target nucleic acid. 
     
     
         212 . The method of  claim 180 , wherein said modifying comprises introducing a single-stranded break in said target nucleic acid. 
     
     
         213 . The method of  claim 180 , wherein said modifying comprises introducing a double-stranded break in said target nucleic acid. 
     
     
         214 . The method of  claim 180 , wherein said modifying comprises allowing said target nucleic acid to be visualized. 
     
     
         215 . The method of  claim 180 , wherein said modifying comprises introducing a modification selected from the group consisting of: an organic dye, a radiolabel, and a spin label, or any combination thereof. 
     
     
         216 . The method of  claim 180 , wherein said modifying comprises deleting a portion of said target nucleic acid. 
     
     
         217 . The method of  claim 180 , wherein said modifying is selected from the group consisting of: activating transcription of said target nucleic acid, and repressing transcription of said target nucleic acid, or any combination thereof. 
     
     
         218 . The method of  claim 180 , wherein said modifying comprises inserting a nucleic acid into said target nucleic acid. 
     
     
         219 . The method of  claim 180 , wherein said eukaryotic cell is an insect cell. 
     
     
         220 . The method of  claim 180 , wherein said eukaryotic cell is a mammalian cell. 
     
     
         221 . The method of  claim 180 , wherein said eukaryotic cell is a mammalian stem cell. 
     
     
         222 . The method of  claim 180 , wherein said eukaryotic cell is a human cell. 
     
     
         223 . The method of  claim 180 , wherein said eukaryotic cell is a human stem cell. 
     
     
         224 . The method of  claim 180 , wherein said eukaryotic cell is a plant cell. 
     
     
         225 . A CRISPR associated polypeptide comprising: an amino acid sequence comprising XXXNHXNNHXXHH, wherein:
 (a) “X” signifies an amino acid identical to an amino acid at the same position in SEQ ID NO: 3, “H” signifies an amino acid similar to an amino acid at the same position in SEQ ID NO: 3, and “N” is an any amino acid; and   (b) said polypeptide comprises an activity selected from the group consisting of: visualizing activity, nuclease activity, or both visualizing and nuclease activity.   
     
     
         226 . The polypeptide of  claim 225 , further comprising at least 18% identity to SEQ ID NO: 3. 
     
     
         227 . The polypeptide of  claim 225 , further comprising at least 17% identity to SEQ ID NO: 4. 
     
     
         228 . The polypeptide of  claim 225  further comprising at least 16% identity to SEQ ID NO: 5. 
     
     
         229 . The polypeptide of  claim 225 , wherein said CRISPR associated polypeptide is adapted to induce a modification in said double-stranded target nucleic acid. 
     
     
         230 . The polypeptide of  claim 229 , wherein said modification is adapted to cleave said double-stranded target nucleic acid. 
     
     
         231 . The polypeptide of  claim 229 , wherein said modification comprises introduction of a single-stranded break in said double-stranded target nucleic acid. 
     
     
         232 . The polypeptide of  claim 229 , wherein said modification comprises introduction of a double-stranded break in said double-stranded target nucleic acid. 
     
     
         233 . The polypeptide of  claim 229 , wherein said modification comprises deletion of a portion of said double-stranded target nucleic acid. 
     
     
         234 . The polypeptide of  claim 229 , wherein said modification comprises introduction of a nucleic acid into said double-stranded target nucleic acid. 
     
     
         235 . The polypeptide of  claim 229 , wherein said modification is selected from the group consisting of: a modification to visualize said double-stranded target nucleic acid, a modification to activate trancription of said double-stranded target nucleic acid, and a modification to repress transcription of said double-stranded target nucleic acid, or any combination thereof. 
     
     
         236 . The polypeptide of  claim 229 , wherein said CRISPR associated polypeptide further comprises a polypeptide selected from the group consisting of: helicase, nuclease, methyltransferase, demthylase, acetylase, deacetylase, phosphatase, kinase, transcription activator, RNA polymerase subunit, transcription repressor, DNA binding polypeptide, DNA structuring polypeptide, marker polypeptide, reporter polypeptide, fluorescent polypeptide, ligand binding polypeptide, signal polypeptide, subcellular localization polypeptide, and antibody epitope, or any combination thereof. 
     
     
         237 . A pharmaceutical composition comprising: the polypeptide of  claim 225 . 
     
     
         238 . A delivery vehicle comprising: the polypeptide of  claim 225 . 
     
     
         239 . The delivery vehicle of  claim 238 , wherein said delivery vehicle is selected from the group consisting of: cationic polymers, lipids, cell-penetrating peptide, and viruses, or any combination thereof. 
     
     
         240 . A kit comprising: the composition of  claim 80 . 
     
     
         241 . A kit comprising: the composition of  claim 109 . 
     
     
         242 . A kit comprising: the vector of  claim 116 . 
     
     
         243 . A kit comprising: the composition of  claim 167 . 
     
     
         244 . A kit comprising: the polypeptide of  claim 225 .