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. Genome engineering can refer to altering the genome by deleting, inserting, mutating, or substituting specific nucleic acid sequences. The altering can be gene or location specific. Genome engineering can use nucleases to cut a nucleic acid thereby generating a site for the alteration. Engineering of non-genomic nucleic acid is also contemplated.
1 . A method of delivering a donor polynucleotide to a target nucleic acid within a nucleus of a cell, the method comprising contacting the cell with a complex comprising:
a. a site-directed polypeptide fused to a reverse transcriptase, and
b. an engineered single guide nucleic acid-targeting nucleic acid (NATNA), the engineered single guide NATNA comprising a spacer that can specifically hybridize to the target nucleic acid, a crRNA, a tracrRNA, and a 3′ hybridizing extension comprising a reverse transcription template and a binding site for the reverse transcriptase, wherein said 3′ hybridizing extension is RNA,
wherein the site-directed polypeptide introduces a single-stranded break within the target nucleic acid, and wherein the reverse transcription template is reverse transcribed by the reverse transcriptase within the nucleus to deliver a newly transcribed DNA comprising the donor polynucleotide to the nucleus of the cell.
2 . The method of claim 1 , wherein the site-directed polypeptide comprises a nuclear localization signal.
3 . The method of claim 1 , wherein the tracrRNA sequence comprises a minimum tracrRNA sequence.
4 . The method of claim 3 , wherein the tracrRNA sequence comprises a minimum tracrRNA sequence followed by a mid-tracrRNA.
5 . The method of claim 4 , wherein the NATNA further comprises a region of hybridization between a minimum CRISPR repeat sequence and a minimum tracrRNA sequence, wherein the region of hybridization forms a first duplex region.
6 . The method of claim 1 , wherein the tracrRNA sequence has at least 60% identity to the S. pyogenes tracrRNA.
7 . The method of claim 1 , wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) in the target nucleic acid.
8 . The method of claim 7 , wherein the protospacer adjacent motif (PAM) consists of a sequence selected from 5′-NGG-3′, 5′-NGGNG-3′, 5′-NNAAAAW-3′, 5′-NNNNGATT-3′, 5′-GNNNCNNA-3′, and 5′-NNNACA-3′.
9 . The method of claim 1 , wherein the site-directed polypeptide is a Cas9 nuclease having at least one substantially inactive nuclease domain.
10 . The method of claim 9 , wherein the site-directed polypeptide is a Cas9 nuclease having a mutation at one or more of the sites Asp10, His840, Asn854 and Asn856.
11 . The method of claim 9 , wherein the site-directed polypeptide is a Cas9 nuclease having one or more mutations selected from D10A, H840A, N854A or N856A.
12 . The method of claim 9 , wherein the site-directed polypeptide is an S. pyogenes Cas9 having at least one substantially inactive nuclease domain.
13 . The method of claim 1 , wherein the reverse transcriptase is selected from an HIV reverse transcriptase, and an MMLV reverse transcriptase.
14 . The method of claim 1 , wherein the method is performed in an isolated cell.
15 . The method of claim 14 , wherein the method results in: 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 the cell, metabolic changes in the cell, or structural changes in the cell.