# US 9,518,258 B2 · Targeted alteration of DNA with oligonucleotides > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,518,258 B2 - Document ID: US9518258B2 - Status: Granted - Kind: B2 - Application number: 13/991,065 - Filed: 2011-11-25 - Published: 2013-12-12 - Granted: 2016-12-13 - Earliest claimed: 2010-12-02 - Assignees: Keygene N.V. - Applicants: Michiel Theodoor Jan De Both; Tomoyuki Furukawa - Inventors: Michiel Theodoor Jan De Both; Tomoyuki Furukawa - Web page: https://patentgrep.com/patent/US9518258B2 ## CPC - C12N 15/1024: {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} - C12N 15/102: {Mutagenizing nucleic acids} - C12N 15/1082: {Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) 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. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 4) 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. ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 6) 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. ### Claim 8 (depends on 6) The method according to claim 6, wherein the first oligonucleotide and/or the second oligonucleotide comprises at least two modified nucleotides. ### Claim 9 (depends on 1) 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. ### Claim 10 (depends on 6) 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. ### Claim 11 (depends on 1) 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. ### Claim 12 (depends on 1) 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. ### Claim 13 (depends on 1) The method according to claim 1, wherein the alteration is a deletion, a substitution and/or an insertion of at least one nucleotide. ### Claim 14 (depends on 1) 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. ### Claim 15 (depends on 1) 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. ### Claim 16 (depends on 2) 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. ### Claim 17 (depends on 2) 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. ### Claim 18 (depends on 6) 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. ### Claim 19 (depends on 8) The method according to claim 8, wherein the first oligonucleotide and/or the second oligonucleotide comprises at least three modified nucleotides. ### Claim 20 (depends on 8) The method according to claim 8, wherein the first oligonucleotide and/or the second oligonucleotide comprises at least four modified nucleotides. ### Claim 21 (depends on 8) The method according to claim 8, wherein the first oligonucleotide and/or the second oligonucleotide comprises at least five modified nucleotides. ### Claim 22 (depends on 8) The method according to claim 8, wherein the first oligonucleotide and/or the second oligonucleotide comprises three modified nucleotides. ### Claim 23 (depends on 8) The method according to claim 8, wherein the first oligonucleotide and/or the second oligonucleotide comprises two, three, four, or five modified nucleotides. ### Claim 24 (depends on 10) 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. ### Claim 25 (depends on 10) The method according to claim 10, wherein the at least one mismatch is located at the 3′ end of said oligonucleotide.