Targeted alteration of DNA with oligonucleotides

Patent metadata

Field	Value
Publication number	US-9518258-B2
Application number	US-201113991065-A
Country	US
Kind code	B2
Filing date	Nov 25, 2011
Priority date	Dec 2, 2010
Publication date	Dec 13, 2016
Grant date	Dec 13, 2016

Abstract

The current invention relates to a method for targeted alteration of acceptor DNA, for example duplex acceptor DNA. The method comprises use of at least two oligonucleotides, each oligonucleotide having at least one mismatch relative to the targeted (duplex) acceptor DNA. The mismatch of the first oligonucleotide is directed to a nucleotide at a position in the first strand of the duplex and the mismatch of the second oligonucleotide is directed to the nucleotide in the second strand that occupies the complementary position in the duplex acceptor DNA (e.g. forms a base-pair with the nucleotide in the first strand). These mismatches are located at specific positions within said oligonucleotides. Also provided is a kit that comprises instructions for performing the method according to the inventions, and in a preferred embodiment, comprises oligonucleotides suitable for use in the method.

First claim

The invention claimed is:

1. A method for targeted alteration of a duplex acceptor DNA sequence comprising the step of
combining the duplex acceptor DNA sequence with at least a first oligonucleotide and a second oligonucleotide, wherein the duplex acceptor DNA sequence comprises a first DNA sequence and a second DNA sequence, which is complementary to the first DNA sequence; and
wherein the first oligonucleotide comprises at least one domain that is capable of hybridizing to the first DNA sequence and wherein the first oligonucleotide further comprises at least one mismatch with respect to the first DNA sequence and wherein this at least one mismatch is positioned at most 2 nucleotides from the 3′ end of said first oligonucleotide;
and wherein the second oligonucleotide comprises at least one domain that is capable of hybridizing to the second DNA sequence and wherein the second oligonucleotide further comprises at least one mismatch with respect to the second DNA sequence and wherein this at least one mismatch is positioned at most 2 nucleotides from the 3′ end of said second oligonucleotide;
and wherein the at least one mismatch in the first oligonucleotide is relative to a nucleotide in the first DNA sequence of the duplex acceptor DNA sequence and wherein the at least one mismatch in the second oligonucleotide is relative to a nucleotide in the second DNA sequence of the duplex acceptor DNA sequence, and wherein said nucleotide in the first DNA sequence and said nucleotide in the second DNA sequence occupy complementary positions in the duplex acceptor DNA sequence;
and wherein the alteration of the duplex acceptor DNA sequence is within a cell.

2. The method according to claim 1 , wherein the mismatch in the first oligonucleotide is positioned at most 1 nucleotide from the 3′ end of said first oligonucleotide and, independently, the mismatch in the second oligonucleotide is positioned at most 1 nucleotide from the 3′ end of said second oligonucleotide.

3. The method according to claim 1 , wherein the domain in the first oligonucleotide comprises or is directly adjacent to the at least one mismatch in the first oligonucleotide and/or the domain in the second oligonucleotide comprises or is directly adjacent to the at least one mismatch in the second oligonucleotide.

4. The method according to claim 1 , wherein the first oligonucleotide is complementary to the first DNA sequence except for the one mismatch and/or wherein the second oligonucleotide is complementary to the second DNA sequence except for the one mismatch.

5. The method according to claim 4 , wherein the mismatch in the first oligonucleotide is at the 3′ end and wherein the mismatch in the second oligonucleotide is at the 3′ end.

6. The method according to claim 1 , wherein the first oligonucleotide comprises at least one section that contains at least one modified nucleotide and/or the second oligonucleotide comprises at least one section that contains at least one modified nucleotide, wherein each said modified nucleotide contains a modification that is independently selected from the group consisting of a base modification, a backbone modification or a sugar modification.

7. The method according to claim 6 , wherein each said modified nucleotide is independently selected from the group consisting of Locked Nucleic Acids (LNA) or phosphorothioate bonds.

8. The method according to claim 6 , wherein the first oligonucleotide and/or the second oligonucleotide comprises at least two modified nucleotides.

9. The method according to claim 1 , wherein the at least one mismatch in the first oligonucleotide and the at least one mismatch in the second oligonucleotide are not modified nucleotides.

10. The method according to claim 6 , wherein the at least one modified nucleotide is at least one nucleotide from the at least one mismatch, and wherein the at least one mismatch is located at most 2 nucleotides from the 3′ end of said oligonucleotide.

11. The method according to claim 1 , wherein the alteration of the duplex acceptor DNA sequence is within a cell selected from the group consisting of a prokaryotic cell, a bacterial cell, a eukaryotic cell, a plant cell, an animal cell, a yeast cell, a fungal cell, a rodent cell, a human cell, a non-human cell, and an embryonic cell.

12. The method according to claim 1 , wherein the duplex acceptor DNA sequence is from a prokaryotic organism, a bacterium, an eukaryotic organism, a plant, an animal, a yeast, a fungus, a rodent, or a human.

13. The method according to claim 1 , wherein the alteration is a deletion, a substitution and/or an insertion of at least one nucleotide.

14. The method according to claim 1 , wherein the duplex acceptor DNA sequence is from genomic DNA, linear DNA, artificial chromosomes, mammalian artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, plant artificial chromosomes, nuclear chromosomal DNA, organellar DNA, plasmid DNA or episomal DNA.

15. The method according to claim 1 , wherein the method is useful for altering a cell, correcting a mutation by restoration to wild type, inducing a mutation, inactivating an enzyme by disruption of coding region, modifying bioactivity of an enzyme by altering coding region, modifying a protein by disrupting the coding region, mismatch repair, targeted alteration of plant genetic material, including gene mutation, targeted gene repair and gene knockout.

16. The method according to claim 2 , wherein the mismatch in the first oligonucleotide is positioned at the 3′ end of said first oligonucleotide or the mismatch in the second oligonucleotide is positioned at the 3′ end of said second oligonucleotide.

17. The method according to claim 2 , wherein the mismatch in the first oligonucleotide is positioned at the 3′ end of said first oligonucleotide and the mismatch in the second oligonucleotide is positioned at the 3′ end of said second oligonucleotide.

18. The method according to claim 6 , wherein at least one modified nucleotide contains a base modification, wherein said base modification is a 3′ or 5′ end base modification.

19. The method according to claim 8 , wherein the first oligonucleotide and/or the second oligonucleotide comprises at least three modified nucleotides.

20. The method according to claim 8 , wherein the first oligonucleotide and/or the second oligonucleotide comprises at least four modified nucleotides.

21. The method according to claim 8 , wherein the first oligonucleotide and/or the second oligonucleotide comprises at least five modified nucleotides.

22. The method according to claim 8 , wherein the first oligonucleotide and/or the second oligonucleotide comprises three modified nucleotides.

23. The method according to claim 8 , wherein the first oligonucleotide and/or the second oligonucleotide comprises two, three, four, or five modified nucleotides.

24. The method according to claim 10 , wherein the at least one mismatch is located at most 1 nucleotide from the 3′ end of said oligonucleotide.

25. The method according to claim 10 , wherein the at least one mismatch is located at the 3′ end of said oligonucleotide.

Assignees

• De Both Michiel Theodoor Jan
• Furukawa Tomoyuki
• Keygene Nv

Inventors

• De Both Michiel Theodoor Jan
• Furukawa Tomoyuki

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/1024 Primary

  In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair · CPC title

• C12N15/1082

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title