GENOME MODIFICATION USING GUIDE POLYNUCLEOTIDE/CAS ENDONUCLEASE SYSTEMS AND METHODS OF USE

• US 20150059010A1
• Filed: 08/20/2014
• Published: 02/26/2015
• Est. Priority Date: 08/22/2013
• Status: Abandoned Application

• Current Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Original Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Inventors: Cigan, Andrew Mark, Patten, Phillip A., Young, Joshua K.
• Application Number:
  US14/463,691
• Publication Number:
  US 20150059010A1
• Time in Patent Office: Days
• US Class Current: 800/260
• CPC Class Codes: A01H 1/00 Processes for modifying genotypes ; Plants characterised by associated natural traits A01H4/00 takes precedence
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8241 Phenotypically and genetically modified plants via recombinant DNA technology
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3533 Halogen

Abstract

Compositions and methods are provided for genome modification of a target sequence in the genome of a cell. The methods and compositions employ a guide polynucleotide/Cas endonuclease system to provide an effective system for modifying or altering target sites within the genome of a cell or organism. Once a genomic target site is identified, a variety of methods can be employed to further modify the target sites such that they contain a variety of polynucleotides of interest. Compositions and methods are also provided for editing a nucleotide sequence in the genome of a cell. Breeding methods and methods for selecting plants utilizing a two component RNA polynucleotide and Cas endonuclease system are also disclosed.

Claims

1. A guide polynucleotide comprising:

(i) a first nucleotide sequence domain that is complementary to a nucleotide sequence in a target DNA; and

,

(ii) a second nucleotide sequence domain that interacts with a Cas endonuclease,

wherein the first nucleotide sequence domain and the second nucleotide sequence domain are composed of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or a combination thereof, wherein the guide polynucleotide does not solely comprise ribonucleic acids.

2. The guide polynucleotide of claim 1, wherein the first nucleotide sequence domain and the second nucleotide sequence domain are located on a single molecule.

3. The guide polynucleotide of claim 1, wherein the second nucleotide sequence domain comprises two separate molecules that are capable of hybridizing along a region of complementarity.

4. The guide polynucleotide of claim 1, wherein the first nucleotide sequence domain is a DNA sequence and the second nucleotide sequence domain is selected from the group consisting of a DNA sequence, a RNA sequence, and a combination thereof.

5. The guide polynucleotide of claim 1, wherein the first nucleotide sequence domain and the second nucleotide sequence domain are DNA sequences.

6. The guide polynucleotide of claim 1, wherein the first nucleotide sequence domain and/or the second nucleotide sequence domain comprises at least one modification, wherein said at least one modification is selected from the group consisting of a 5′

cap, a 3′

polyadenylated tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide poly nucleotide to a subcellular location, a modification or sequence that provides for tracking, a modification or sequence that provides a binding site for proteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a 2,6-Diaminopurine nucleotide, a 2′

-Fluoro A nucleotide, a 2′

-Fluoro U nucleotide;

a 2′

-O-Methyl RNA nucleotide, a phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 molecule, a 5′

to 3′

covalent linkage, and any combination thereof.

7. The guide polynucleotide of claim 1, wherein the first nucleotide sequence domain and/or the second nucleotide sequence domain comprises at least one modification that provides for an additional beneficial feature, wherein said at least one modification is selected from the group consisting of a 5′

cap, a 3′

polyadenylated tail, a riboswitch sequence, a stability control sequence;

a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide poly nucleotide to a subcellular location, a modification or sequence that provides for tracking, a modification or sequence that provides a binding site for proteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a 2,6-Diaminopurine nucleotide, a 2′

-Fluoro A nucleotide, a 2′

-Fluoro U nucleotide;

a 2′

-O-Methyl RNA nucleotide, a phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 molecule, a 5′

to 3′

covalent linkage, and any combination thereof.

8. The guide polynucleotide of claim 7, wherein the additional beneficial feature is selected from the group consisting of a modified or regulated stability, a subcellular targeting, tracking, a fluorescent label, a binding site for a protein or protein complex, modified binding affinity to complementary target sequence, modified resistance to cellular degradation, and increased cellular permeability.

9. A plant or seed comprising the guide polynucleotide of claim 1.

10. A guide polynucleotide/Cas endonuclease complex wherein the guide polynucleotide comprises:

(i) a first nucleotide sequence domain that is complementary to a nucleotide sequence in a target DNA; and

,

(ii) a second nucleotide sequence domain that interacts with a Cas endonuclease, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein said guide polynucleotide and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site.

11. The guide polynucleotide/Cas endonuclease complex of claim 10, wherein the first nucleotide sequence domain and the second nucleotide sequence domain of the guide polynucleotide are composed of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or a combination thereof, wherein the guide polynucleotide does not solely comprise ribonucleic acids.

12. The guide polynucleotide/Cas endonuclease complex of claim 10, wherein the first nucleotide sequence domain and/or the second nucleotide sequence domain of said guide polynucleotide comprises at least one modification that provides for an additional beneficial feature, wherein said at least one modification is selected from the group consisting of a 5′

cap, a 3′

polyadenylated tail, a riboswitch sequence, a stability control sequence;

a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide poly nucleotide to a subcellular location, a modification or sequence that provides for tracking, a modification or sequence that provides a binding site for proteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a 2,6-Diaminopurine nucleotide, a 2′

-Fluoro A nucleotide, a 2′

-Fluoro U nucleotide;

a 2′

-O-Methyl RNA nucleotide, a phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 molecule, a 5′

to 3′

covalent linkage, and any combination thereof.

13. The guide polynucleotide/Cas endonuclease complex of claim 10, wherein the Cas endonuclease is a Cas9 endonuclease.

14. A plant or seed comprising the guide polynucleotide/Cas endonuclease complex of claims 10.

15. A method for modifying a target site in the genome of a cell, the method comprising providing a guide polynucleotide to a cell having a Cas endonuclease, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein said guide polynucleotide and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site.

16. A method for modifying a target site in the genome of a cell, the method comprising providing a guide polynucleotide and a Cas endonuclease to a cell, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein said guide polynucleotide and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site.

17. The method of claim 16, further comprising providing a donor DNA to said cell, wherein said donor DNA comprises a polynucleotide of interest.

18. The method of any one of claims 16, further comprising identifying at least one cell that has a modification at said target, wherein the modification at said target site is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).

21. The method of claims 16, wherein the guide polynucleotide is provided directly by particle bombardment.

22. The method of claims 16, wherein the guide polynucleotide is provided via particle bombardment or Agrobacterium transformation of a recombinant DNA construct comprising a U6 polymerase III promoter.

23. The method of claims 16, wherein the guide polynucleotide is a single guide polynucleotide comprising a variable targeting domain and a cas endonuclease recognition domain.

24. The method of claims 16, wherein the guide polynucleotide is a duplex guide polynucleotide comprising a crNucleotide molecule and a tracrNucleotide molecule.

29. The method of claim 16, wherein the cell is selected from the group consisting of a non-human animal, bacterial, fungal, insect, yeast, and a plant cell.

30. The method of claim 29, wherein the plant cell is selected from the group consisting of a monocot and dicot cell.

31. The method of claim 29, wherein the plant cell is selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tobacco, Arabidopsis, and safflower cell.

38. The plant or plant cell of claim 29 wherein the at least one nucleotide modification is not a modification at said target site.

19. A method for introducing a polynucleotide of interest into a target site in the genome of a cell, the method comprising:

a) providing a guide polynucleotide, a donor DNA and a Cas endonuclease to a cell, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein said guide polynucleotide and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site;

b) contacting the cell of (a) with a donor DNA comprising a polynucleotide of interest; and

,

c) identifying at least one cell from (b) comprising in its genome the polynucleotide of interest integrated at said target site.

20. The method of claim 19, wherein the donor DNA and Cas endonuclease are introduced into said cell using at least one recombinant DNA construct capable of expressing the donor DNA and/or the Cas endonuclease.

25. A method for modifying a target site in the genome of a cell, the method comprising:

a) providing to a cell a crNucleotide, a first recombinant DNA construct capable of expressing a tracrRNA, and a second recombinant DNA capable of expressing a Cas endonuclease, wherein said crNucleotide is a deoxyribonucleotide sequence or a combination of a deoxyribonucleotide and ribonucleotide sequence, wherein said crNucleotide, said tracrRNA and said Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site; and

,

b) identifying at least one cell that has a modification at said target site, wherein the modification is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).

26. A method for modifying a target site in the genome of a cell, the method comprising:

a) providing to a cell a tracrNucleotide, a first recombinant DNA construct capable of expressing a crRNA and a second recombinant DNA capable of expressing a Cas endonuclease, wherein said tracrNucleotide is selected a deoxyribonucleotide sequence or a combination of a deoxyribonucleotide and ribonucleotide sequence, wherein said tracrNucleotide, said crRNA and said Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site; and

,

b) identifying at least one cell that has a modification at said target site, wherein the modification is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).

27. A method for introducing a polynucleotide of interest into a target site in the genome of a cell, the method comprising:

a) providing to a cell a first recombinant DNA construct capable of expressing a guide polynucleotide, and a second recombinant DNA construct capable of expressing a Cas endonuclease, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein said guide polynucleotide and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site;

b) contacting the cell of (a) with a donor DNA comprising a polynucleotide of interest; and

,

c) identifying at least one cell from (b) comprising in its genome the polynucleotide of interest integrated at said target site.

28. A method for editing a nucleotide sequence in the genome of a cell, the method comprising introducing a guide polynucleotide, a polynucleotide modification template and at least one Cas endonuclease into a cell, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein the Cas endonuclease introduces a double-strand break at a target site in the genome of said cell, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence.

32. A plant or seed comprising a guide polynucleotide and a Cas9 endonuclease, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein said Cas9 endonuclease and guide polynucleotide are capable of forming a complex and creating a double strand break in a genomic target site of said plant.

39. The plant of claim 32, wherein the plant is a monocot or a dicot.

40. The plant of claim 39, wherein the monocot is selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass.

41. The plant of claim 39, wherein the dicot is selected from the group consisting of soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tobacco, Arabidopsis, or safflower.

33. A plant or seed comprising a recombinant DNA construct and a guide polynucleotide, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein said recombinant DNA construct comprises a promoter operably linked to a nucleotide sequence encoding a plant optimized Cas endonuclease, wherein said plant optimized Cas endonuclease and guide polynucleotide are capable of forming a complex and creating a double strand break in a genomic target site of said plant.

34. The plant of claim 33, further comprising a polynucleotide of interest integrated into said genomic target site of said plant.

35. The plant or seed of claim 33 further comprising a modification at said genomic target site, wherein the modification is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).

36. A plant or seed comprising at least one altered target sequence, wherein the at least one altered target sequence originated from a corresponding target sequence that was recognized and cleaved by a guide polynucleotide/Cas endonuclease complex, wherein the Cas endonuclease is capable of introducing a double-strand break at said target site in the plant genome, wherein said guide polynucleotide does not solely comprise ribonucleic acids.

37. A plant or seed comprising a modified nucleotide sequence, wherein the modified nucleotide sequence was produced by providing a guide polynucleotide, a polynucleotide modification template and at least one Cas endonuclease to a cell, wherein said guide polynucleotide does not solely comprise ribonucleic acids, wherein the Cas endonuclease is capable of introducing a double-strand break at a target site in the plant genome, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence.

42. A method for selecting a plant comprising an altered target site in its plant genome, the method comprising:

a) obtaining a first plant comprising at least one Cas endonuclease capable of introducing a double strand break at a target site in the plant genome;

b) obtaining a second plant comprising a guide polynucleotide that is capable of forming a complex with the Cas endonuclease of (a), wherein the guide polynucleotide does not solely comprise ribonucleic acids, c) crossing the first plant of (a) with the second plant of (b);

d) evaluating the progeny of (c) for an alteration in the target site and e) selecting a progeny plant that possesses the desired alteration of said target site.

43. A method for selecting a plant comprising an altered target site in its plant genome, the method comprising:

a) obtaining a first plant comprising at least one Cas endonuclease capable of introducing a double strand break at a target site in the plant genome;

b) obtaining a second plant comprising a guide polynucleotide and a donor DNA, wherein the guide polynucleotide does not solely comprise ribonucleic acids, wherein said guide polynucleotide is capable of forming a complex with the Cas endonuclease of (a), wherein said donor DNA comprises a polynucleotide of interest;

c) crossing the first plant of (a) with the second plant of (b);

d) evaluating the progeny of (c) for an alteration in the target site and e) selecting a progeny plant that comprises the polynucleotide of interest inserted at said target site.