CRISPR hybrid DNA/RNA polynucleotides and methods of use

Patent metadata

Field	Value
Publication number	US-9868962-B2
Application number	US-201715679555-A
Country	US
Kind code	B2
Filing date	Aug 17, 2017
Priority date	Jan 28, 2015
Publication date	Jan 16, 2018
Grant date	Jan 16, 2018

Abstract

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.

First claim

What is claimed is:

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 se

Assignees

• Pioneer Hi Bred Int
• Pioneer Hi Bred Int

Inventors

• May Andrew Paul
• Donohoue Paul Daniel

Classifications

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12N15/111 Primary

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N15/8213

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title