MODIFIED CASCADE RIBONUCLEOPROTEINS AND USES THEREOF
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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.
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Citations
244 Claims
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1-46. -46. (canceled)
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47. A composition comprising:
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(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. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)
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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. - View Dependent Claims (81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 240)
- a nucleic acid-targeting nucleic acid comprising;
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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.
- View Dependent Claims (110, 111, 112, 113, 114, 115, 241)
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116. A vector comprising:
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(a) a first polynucleotide sequence encoding a CRISPR associated polypeptide; and (b) a second polynucleotide sequence encoding a visualizing polypeptide. - View Dependent Claims (117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 242)
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129. A method for modifying a genomic target nucleic acid in a eukaryotic cell comprising:
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(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. - View Dependent Claims (130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166)
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167. A composition comprising:
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(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. - View Dependent Claims (168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 243)
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180. A method for using a eukaryotic cell comprising:
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(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. - View Dependent Claims (181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224)
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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. - View Dependent Claims (226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 244)
- an amino acid sequence comprising XXXNHXNNHXXHH, wherein;
Specification