Compositions and methods for modifying genomes

• US 9,896,696 B2
• Filed: 02/14/2017
• Issued: 02/20/2018
• Est. Priority Date: 02/15/2016
• Status: Active Grant

• Current Assignee: RiceTec, Inc.
• Original Assignee: Benson Hill Biosystems, Inc.
• Inventors: Begemann, Matthew, Gray, Benjamin Neil
• Primary Examiner(s): Page, Brent T
• Application Number:
  US15/432,109
• Publication Number:
  US 20170233756A1
• Time in Patent Office: 371 Days
• Field of Search: None
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/8202 by biological means, e.g. cell mediated or natural vector
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Compositions and methods for modifying genomic DNA sequences are provided. The methods produce double-stranded breaks (DSBs) at pre-determined target sites in a genomic DNA sequence, resulting in mutation, insertion, and/or deletion of DNA sequences at the target site(s) in a genome. Compositions comprise DNA constructs comprising nucleotide sequences that encode a Cpf1 or Csm1 protein operably linked to a promoter that is operable in the cells of interest. The DNA constructs can be used to direct the modification of genomic DNA at pre-determined genomic loci. Methods to use these DNA constructs to modify genomic DNA sequences are described herein. Additionally, compositions and methods for modulating the expression of genes are provided. Compositions comprise DNA constructs comprising a promoter that is operable in the cells of interest operably linked to nucleotide sequences that encode a mutated Cpf1 or Csm1 protein with an abolished ability to produce DSBs, optionally linked to a domain that regulates transcriptional activity. The methods can be used to up- or down-regulate the expression of genes at predetermined genomic loci.

Claims

1. A method of modifying a nucleotide sequence at a target site in the genome of a eukaryotic cell comprising:

introducing into said eukaryotic cell

(i) a DNA-targeting RNA, or a DNA polynucleotide encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises;

(a) a first segment comprising a nucleotide sequence that is complementary to a sequence in the target DNA; and

(b) a second segment that interacts with a Csm1 polypeptide; and

(ii) a Csm1 polypeptide, or a polynucleotide encoding a Csm1 polypeptide, wherein the Csm1 polypeptide comprises;

(a) an RNA-binding portion that interacts with the DNA-targeting RNA; and

(b) an activity portion that exhibits site-directed enzymatic activity,

wherein said Csm1 polypeptide has at least 95% identity with a sequence selected from the group consisting of;

SEQ ID NOs;

134, 147, 160, and 230, and has Csm1 nuclease activity, wherein said method modifies said nucleotide sequence at said target site, and wherein said genome of a eukaryotic cell is a nuclear, plastid, or mitochondrial genome.

3. The method of claim 1 wherein said eukaryotic cell is a plant cell.

4. The method of claim 3, further comprising:

culturing the plant cell to produce a plant under conditions in which the Csm1 polypeptide is expressed and cleaves the nucleotide sequence at the target site to produce a modified nucleotide sequence; and

selecting a plant comprising said modified nucleotide sequence.

5. The method of claim 1, wherein said Csm1 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:

134, 147, 160, and 230.

7. The method of claim 1, wherein said modified nucleotide sequence comprises insertion of heterologous DNA into the genome of the cell, deletion of a nucleotide sequence from the genome of the cell, or mutation of at least one nucleotide in the genome of the cell.

9. The method of claim 1 wherein said modified nucleotide sequence comprises insertion of a polynucleotide that encodes a protein conferring antibiotic or herbicide tolerance to transformed cells.

10. The method of claim 9 wherein said polynucleotide that encodes a protein conferring antibiotic or herbicide tolerance comprises SEQ ID NO:

76, or encodes a protein that comprises SEQ ID NO;

77.

16. A eukaryotic cell produced by the method of claim 1.

17. The eukaryotic cell of claim 16 wherein said eukaryotic cell is a plant cell.

20. A plant produced by the method of claim 3.

22. The seed of the plant of claim 20.

24. The method of claim 3 wherein said polynucleotide encoding a Csm1 polypeptide is codon-optimized for expression in a plant cell.

2. A method of modifying a nucleotide sequence at a target site in the genome of a prokaryotic cell comprising:

introducing into said prokaryotic cell

(i) a DNA-targeting RNA, or a DNA polynucleotide encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises;

(a) a first segment comprising a nucleotide sequence that is complementary to a sequence in the target DNA; and

(b) a second segment that interacts with a Csm1 polypeptide; and

(ii) a Csm1 polypeptide, or a polynucleotide encoding a Csm1 polypeptide, wherein the Csm1 polypeptide comprises;

(a) an RNA-binding portion that interacts with the DNA-targeting RNA; and

(b) an activity portion that exhibits site-directed enzymatic activity,

wherein said Csm1 polypeptide has at least 95% identity with a sequence selected from the group consisting of;

SEQ ID NOs;

134, 147, 160, and 230 and has Csm1 nuclease activity, wherein said method modifies said nucleotide sequence at said target site, wherein said genome of a prokaryotic cell is a chromosomal, plasmid, or other intracellular DNA sequence, and wherein said prokaryotic cell is not the natural host of a gene encoding said Csm1 polypeptide.

6. The method of claim 2, wherein said Csm1 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:

134, 147, 160, and 230.

8. The method of claim 2, wherein said modified nucleotide sequence comprises insertion of heterologous DNA into the genome of the cell, deletion of a nucleotide sequence from the genome of the cell, or mutation of at least one nucleotide in the genome of the cell.

11. A nucleic acid molecule comprising a polynucleotide sequence encoding a Csm1 polypeptide, wherein said polynucleotide sequence has at least 95% identity with a sequence selected from the group consisting of SEQ ID NOs:

185, 186, and 193, or a fragment or variant thereof, or wherein said polynucleotide sequence encodes a Csm1 polypeptide with at least 95% identity to a sequence selected from the group consisting of SEQ ID NOs;

134, 147, 160, and 230 and has Csm1 nuclease activity, and wherein said polynucleotide sequence encoding a Csm1 polypeptide is operably linked to a promoter that is heterologous to the polynucleotide sequence encoding a Csm1 polypeptide.

12. The nucleic acid molecule of claim 11 wherein said nucleic acid molecule comprising a polynucleotide sequence encoding a Csm1 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs:

185, 186, and 193, or encodes a Csm1 polypeptide that comprises a sequence selected from the group consisting of SEQ ID NOs;

134, 147, 160, and 230.

13. A Csm1 polypeptide encoded by the nucleic acid molecule of claim 11.

14. A plant cell, eukaryotic cell, or prokaryotic cell comprising the nucleic acid molecule of claim 11.

15. A plant cell, eukaryotic cell, or prokaryotic cell comprising the nucleic acid molecule of claim 12.

18. A plant comprising the nucleic acid molecule of claim 11.

19. A plant comprising the nucleic acid molecule of claim 12.

21. The seed of the plant of claim 18.

23. The nucleic acid molecule of claim 11 wherein said polynucleotide sequence encoding a Csm1 polypeptide is codon-optimized for expression in a plant cell.