CRISPR hybrid DNA/RNA polynucleotides and methods of use
The present disclosure provides DNA-guided CRISPR systems; polynucleotides comprising DNA, RNA and mixtures thereof for use with CRISPR systems; and methods of use involving such polynucleotides and DNA-guided CRISPR systems.
1. A set of two Class 2 CRISPR polynucleotides comprising:
(i) a first Class 2 CRISPR polynucleotide wherein the first Class 2 CRISPR polynucleotide comprises a targeting region comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and an activating region adjacent to said targeting region; and,
(ii) a second Class 2 CRISPR polynucleotide wherein the second Class 2 CRISPR polynucleotide comprises an activating region wherein the activating region comprises DNA and a sequence that is complementary to a sequence in said activating region of the first Class 2 CRISPR polynucleotide, wherein said activating region of the first Class 2 CRISPR polynucleotide and said activating region of the second Class 2 CRISPR polynucleotide are capable of hybridizing to each other to form an activating duplex region, wherein said activating duplex region comprises a stem and a bulge, and wherein said activating duplex region is capable of binding with a Cas9.
2. The set of two Class 2 CRISPR polynucleotides of claim 1 , wherein said targeting region of the first Class 2 CRISPR polynucleotide comprises a mixture of DNA and RNA.
3. The set of two Class 2 CRISPR polynucleotides of claim 1 , wherein said activating duplex region comprises a compound selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, amino alkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates.
4. The set of two Class 2 CRISPR polynucleotides of claim 1 , wherein said activating duplex region comprises a lower stem, a bulge, and an upper stem.
5. A Class 2 CRISPR system consisting essentially of:
(i) a set of two Class 2 CRISPR polynucleotides comprising (a) a first Class 2 CRISPR polynucleotide wherein the first Class 2 CRISPR polynucleotide comprises a targeting region comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and an activating region adjacent to said targeting region; and (b) a second Class 2 CRISPR polynucleotide wherein the second Class 2 CRISPR polynucleotide comprises an activating region wherein the activating region comprises DNA and a sequence that is complementary to a sequence in said activating region of the first Class 2 CRISPR polynucleotide, wherein said activating region of the first Class 2 CRISPR polynucleotide and said activating region of the second Class 2 CRISPR polynucleotide are capable of hybridizing to each other to form an activating duplex region, wherein said activating duplex region comprises a stem and a bulge, and wherein said activating duplex region is capable of binding with a Cas9; and
(ii) a Cas9.
6. The Class 2 CRISPR system of claim 5 , wherein said activating duplex region comprises a lower stem, a bulge, and an upper stem.
7. The Class 2 CRISPR system of claim 5 , wherein said targeting region of the first Class 2 CRISPR polynucleotide comprises a mixture of DNA and RNA.
8. The Class 2 CRISPR system of claim 5 , wherein said activating duplex region comprises a mixture of DNA and RNA.
9. The Class 2 CRISPR system of claim 5 , wherein said activating duplex region comprises a compound selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, amino alkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates.
10. The Class 2 CRISPR system of claim 5 , further comprising a donor polynucleotide.
11. A method of modifying a target nucleic acid molecule in a non-human organism, an isolated cell, or in vitro, wherein said method comprises:
introducing into a cell
(i) a set of two Class 2 CRISPR polynucleotides comprising (a) a first Class 2 CRISPR polynucleotide wherein the first Class 2 CRISPR polynucleotide comprises a targeting region configured to hybridize to a target sequence within the target nucleic acid and an activating region adjacent to said targeting region; and (b) a second Class 2 CRISPR polynucleotide wherein the second Class 2 CRISPR polynucleotide comprises an activating region wherein the activating region comprises DNA and a sequence that is complementary to a sequence in said activating region of the first Class 2 CRISPR polynucleotide, wherein said activating region of the first Class 2 CRISPR polynucleotide and said activating region of the second Class 2 CRISPR polynucleotide are capable of hybridizing to each other to form an activating duplex region, and wherein said activating duplex region comprises a stem and a bulge; and
(ii) a Cas9, wherein the Cas9 binds with said activating duplex region of the two Class 2 CRISPR polynucleotides, wherein said targeting region of the first Class 2 CRISPR polynucleotide hybridizes to said target sequence, and wherein said target nucleic acid is cleaved.
12. The method of claim 11 , wherein the Cas9 is encoded by an expression vector comprising a coding sequence for the Cas9.
13. The method of claim 11 , wherein the set of two Class 2 CRISPR polynucleotides and the Cas9 form a nucleoprotein complex prior to introduction into the cell.
14. The method of claim 11 , wherein the Cas9 comprises a nuclear localization signal (NLS).
15. The method of claim 11 , wherein the set of two Class 2 CRISPR polynucleotides and the Cas9 are introduced into the cell by lipofection, electroporation, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation, lipid:nucleic acid conjugates, or combinations thereof.
16. The method of claim 11 , wherein said activating duplex region comprises a compound selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, amino alkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates.
17. The method of claim 11 , wherein said activating duplex region comprises a lower stem, a bulge, and an upper stem.
18. The method of claim 11 , wherein the cell is selected from the group consisting of a bacterial cell, an archaeal cell, a plant cell, an algal cell, a fungal cell, an invertebrate cell, a vertebrate cell, a mammalian cell, and a human cell.
19. The method of claim 11 , wherein the targeting region comprises RNA.
20. The method of claim 11 , further comprising introducing a donor polynucleotide into the cell.
21. A method of modulating transcription of at least one gene within a target nucleic acid molecule in a non-human organism, an isolated cell, or in vitro, wherein said method comprises: introducing into a cell
(i) a set of two Class 2 CRISPR polynucleotides comprising (a) a first Class 2 CRISPR polynucleotide wherein the first Class 2 CRISPR polynucleotide comprises a targeting region configured to hybridize to a target sequence within the open reading frame of the at least one gene or a target sequence within the promoter sequence of the at least one gene and an activating region adjacent to said targeting region; and (b) a second Class 2 CRISPR polynucleotide wherein the second Class 2 CRISPR polynucleotide comprises an activating region wherein the activating region comprises DNA and a sequence that is complementary to a sequence in said activating region of the first Class 2 CRISPR polynucleotide, wherein said activating region of the first Class 2 CRISPR polynucleotide and said activating region of the second Class 2 CRISPR polynucleotide are capable of hybridizing to each other to form an activating duplex region, and wherein said activating duplex region comprises a stem and a bulge; and
(ii) a Cas9, wherein the Cas9 has no nuclease activity, wherein the Cas9 binds with said activating duplex region of the two Class 2 CRISPR polynucleotides, wherein said targeting region of the first Class 2 CRISPR polynucleotide hybridizes to said target sequence, and wherein the transcription of at least one gene within the target nucleic acid molecule is modulated.
22. The method of claim 21 , wherein the Cas9 is encoded by an expression vector comprising a coding sequence for the Cas9.
23. The method of claim 21 , wherein the set of two Class 2 CRISPR polynucleotides and the Cas9 form a nucleoprotein complex prior to introduction into the cell.
24. The method of claim 21 , wherein the Cas9 comprises a nuclear localization signal (NLS).
25. The method of claim 21 , wherein the set of two Class 2 CRISPR polynucleotides and the Cas9 are introduced into the cell by lipofection, electroporation, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation, lipid:nucleic acid conjugates, or combinations thereof.
26. The method of claim 21 , wherein said activating duplex region comprises a compound selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, amino alkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates.
27. The method of claim 21 , wherein said activating duplex region comprises a lower stem, a bulge, and an upper stem.
28. The method of claim 21 , wherein the cell is selected from the group consisting of a bacterial cell, an archaeal cell, a plant cell, an algal cell, a fungal cell, an invertebrate cell, a vertebrate cell, a mammalian cell, and a human cell.
29. The method of claim 21 , wherein said targeting region comprises RNA.