COMPOSITIONS AND METHODS OF NUCLEIC ACID-TARGETING NUCLEIC ACIDS
This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof.
1 .- 26 . (canceled)
27 . A composition comprising:
(a) an engineered nucleic acid-targeting nucleic acid comprising:
(i) a tracrRNA sequence; and
(ii) a 3′ hybridizing extension; and
(b) a donor polynucleotide,
wherein said donor polynucleotide is hybridized to said 3′ hybridizing extension.
28 . The composition of claim 27 , wherein said 3′ hybridizing extension is located at the 3′ end of said engineered nucleic acid-targeting nucleic acid.
29 . The composition of claim 27 , wherein said 3′ hybridizing extension is adapted to hybridize to at least 5 nucleotides from the 3′ end of said donor polynucleotide.
30 . The composition of claim 27 , wherein said 3′ hybridizing extension is adapted to hybridize to at least 5 nucleotides from the 5′ end of said donor polynucleotide.
31 . The composition of claim 27 , wherein said 3′ hybridizing extension is adapted to hybridize to at least 5 adjacent nucleotides within said donor polynucleotide.
32 . The composition of claim 27 , wherein said 3′ hybridizing extension is adapted to hybridize to all of said donor polynucleotide.
33 . The composition of claim 27 , wherein said 3′ hybridizing extension is adapted to hybridize to said donor polynucleotide with one or more mismatches.
34 . The composition of claim 27 , wherein said 3′ hybridizing extension comprises a reverse transcription template.
35 . The composition of claim 34 , wherein said reverse transcription template is adapted to be reverse transcribed by a reverse transcriptase.
36 . The composition of claim 34 , wherein further comprising a reverse transcribed DNA polynucleotide.
37 . The composition of claim 36 , wherein said reverse transcribed DNA polynucleotide is adapted to hybridize to said reverse transcription template.
38 . The composition of claim 27 , wherein said donor polynucleotide is DNA.
39 . The composition of claim 27 , wherein said 3′ hybridizing extension is RNA.
40 . The composition of claim 27 , wherein said tracrRNA sequence comprises at least 80% identity to a tracrRNA from any of SEQ ID NOs: 431-562 over 6 contiguous nucleotides.
41 . The composition of claim 27 , wherein said engineered nucleic acid-targeting nucleic acid further comprises a CRISPR RNA sequence.
42 . The composition of claim 40 , wherein said CRISPR RNA comprises at least 80% identity to a CRISPR RNA from any of SEQ ID NOs: 563-679 over 6 contiguous nucleotides.
43 . The composition of claim 27 , wherein said engineered nucleic acid-targeting nucleic acid is an isolated engineered nucleic acid-targeting nucleic acid.
44 . The composition of claim 41 , wherein said engineered nucleic acid-targeting nucleic acid comprises a linker linking said tracrRNA sequence and said CRISPR RNA sequence.
45 . The composition of claim 27 , wherein said engineered nucleic acid-targeting nucleic acid is a recombinant engineered nucleic acid-targeting nucleic acid.
46 . A method for introducing a donor polynucleotide into a target nucleic acid comprising:
(a) contacting said target nucleic acid with the composition of claim 27 ; and
(b) cleaving said target nucleic acid to produce a cleaved target nucleic acid, wherein said cleaving is performed by a polypeptide comprising at least 75% amino acid identity to a sequence from SEQ ID NOs: 1-256 and 795-1346; and
(c) inserting said donor polynucleotide into said cleaved target nucleic acid.
47 . The method of claim 46 , wherein said cleaving is performed by a polypeptide comprising at least 50% amino acid identity to a nuclease domain of SEQ ID NOs: 1-256 and 795-1346.
48 . The method of claim 46 , further comprising producing a therapeutic outcome.
49 . The method of claim 48 , wherein said therapeutic outcome is modulated by said donor polynucleotide.
50 . The method of claim 48 , wherein said therapeutic outcome is a modulation selected from the group consisting of: a decrease in the levels of a protein in a pathway related to a disease, an increase in the levels of a protein in a pathway related to a disease, morphological changes in a cell, metabolic changes in a cell, and structural changes in a cell, or any combination thereof.
51 . A pharmaceutical composition comprising the composition of claim 27 .
52 . A vector comprising a polynucleotide sequence encoding the engineered nucleic acid-targeting nucleic acid comprising a 3′ hybridizing extension of claim 27
53 . A genetically modified cell comprising the composition of claim 27 .
54 . A kit comprising:
(a) the composition of claim 27 ; and
(b) a buffer.
55 . The kit of claim 54 , further comprising instructions for use.