# US 10,767,173 B2 · Method for converting genome sequence of gram-positive bacterium by specifically converting nucleic acid base of targeted DNA sequence, and molecular complex used in same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,767,173 B2 - Document ID: US10767173B2 - Status: Granted - Kind: B2 - Application number: 15/757,243 - Filed: 2016-09-09 - Published: 2019-07-04 - Granted: 2020-09-08 - Earliest claimed: 2015-09-09 - Assignees: National University Corporation Kobe University; Nippon Shokubai Co., Ltd. - Applicants: National University Corporation Kobe University; Nippon Shokubai Co., Ltd. - Inventors: Masaharu Mukoyama; Eita Ichige; Keiji Nishida; Akihiko Kondo - Web page: https://patentgrep.com/patent/US10767173B2 ## CPC - C12N 15/102: {Mutagenizing nucleic acids} - C07K 14/245: Escherichia (G) - C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} - C12N 1/20: Bacteria; Culture media therefor - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5) - C12N 15/09: Recombinant DNA-technology - C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) - C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor - C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR] - C12Y 305/04005: Cytidine deaminase (3.5.4.5) - Y02E 50/10: Biofuels, e.g. bio-diesel - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12Y: ENZYMES - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract The invention provides a method of modifying a targeted site of gram-positive bacterium of a double stranded DNA. The method includes contacting the double-stranded DNA with a complex of a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a given double stranded DNA and a nucleic acid base converting enzyme to convert, delete, or insert one or more nucleotides in the targeted site without cleaving at least one strand of the double stranded DNA in the targeted site, by introducing the nucleic acid encoding the complex into the gram-positive bacterium. The invention also provide a nucleic acid-modifying enzyme complex of a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a double stranded DNA of a gram-positive bacterium and a nucleic acid base converting enzyme bonded to each other, which complex is used for the method. ## Claims ### Claim 1 A method of modifying a targeted site in a double stranded DNA of a gram-positive bacterium, comprising a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a given double stranded DNA and a deaminase are bonded, with said double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into said targeted site, without cleaving at least one strand of said double stranded DNA in the targeted site, wherein the double stranded DNA is contacted with the complex by introducing the nucleic acid encoding the complex into the gram-positive bacterium, and wherein the gram-positive bacterium is a microorganism other than of the genus Bacillus. ### Claim 2 (depends on 1) The method according to claim 1, wherein said nucleic acid sequence-recognizing module is selected from the group consisting of a CRISPR-Cas system wherein at least one DNA cleavage ability of Cas is inactivated, a zinc finger motif, a TAL effector and a PPR motif. ### Claim 3 (depends on 1) The method according to claim 1, wherein said nucleic acid sequence-recognizing module is a CRISPR-Cas system wherein at least one DNA cleavage ability of Cas is inactivated. ### Claim 4 (depends on 1) The method according to claim 1, which uses two or more kinds of nucleic acid sequence-recognizing modules respectively specifically binding to different target nucleotide sequences. ### Claim 5 (depends on 4) The method according to claim 4, wherein said different target nucleotide sequence is present in a different gene. ### Claim 6 (depends on 1) The method according to claim 1, wherein said deaminase is cytidine deaminase. ### Claim 7 (depends on 1) The method according to claim 1, wherein said gram-positive bacterium is a microorganism belonging to the genus Clostridium, the genus Brevibacillus or the genus Corynebacterium. ### Claim 8 (depends on 7) The method according to claim 7, wherein the microorganism belonging to the genus Clostridium is Clostridium saccharoperbutylacetonicum. ### Claim 9 (depends on 7) The method according to claim 7, wherein the microorganism belonging to the genus Brevibacillus is Brevibacillus choshinensis. ### Claim 10 (depends on 7) The method according to claim 7, wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum. ### Claim 11 (depends on 1) The method according to claim 1, comprising a step of introducing an expression vector comprising a nucleic acid encoding said complex in a form permitting control of an expression period into said gram-positive bacterium, and a step of inducing expression of the nucleic acid for a period necessary for fixing the modification of the targeted site in the double stranded DNA. ### Claim 12 (depends on 6) The method according to claim 6, wherein said cytidine deaminase is PmCDA1.