# US 2020/0399659 A1 · Helper plasmid for transformation, method for producing transformant using the same, and transformation method > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399659 A1 - Document ID: US20200399659A1 - Status: Published application - Kind: A1 - Application number: 16/909,499 - Filed: 2020-06-23 - Published: 2020-12-24 - Earliest claimed: 2019-06-24 - Assignees: Toyota Jidosha Kabushiki Kaisha - Applicants: Toyota Jidosha Kabushiki Kaisha - Inventors: Toru Onishi; Nobuki Tada - Web page: https://patentgrep.com/patent/US20200399659A1 ## CPC - C12N 15/905: {in yeast} - 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) - C12N 15/65: using markers (enzymes used as markers C12N15/52) - C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease - C12N 15/81: for yeasts - C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract A stable transformant, in which a gene of interest is incorporated into the genome, is simply and efficiently produced. A method for producing a transformant, comprising a step of introducing into a host, linear genome-introduced nucleic acid fragment(s) comprising a gene of interest and a helper plasmid for transformation having a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragment(s), and then selecting a transformant, in which the gene of interest is incorporated into the predetermined position in the host genome and the gene of interest is expressed therein. ## Claims ### Claim 1 A method for producing a transformant, comprising steps of: introducing into a host, one or a plurality of linear genome-introduced nucleic acid fragments each comprising a gene of interest to be introduced into a predetermined position on a genome, and a helper plasmid for transformation having a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragments, wherein, in a state in which the linear genome-introduced nucleic acid fragments are incorporated into the helper plasmid for transformation, a pair of homologous recombination sequences for homologous recombination that takes place outside of the gene of interest and at the predetermined position on the genome, and a pair of endonuclease target sequences outside of the pair of homologous recombination sequences are disposed; and selecting a transformant, in which the gene of interest is incorporated in the predetermined position on the host genome and the gene of interest is expressed therein. ### Claim 2 (depends on 1) The method for producing a transformant according to claim 1, wherein the helper plasmid for transformation comprises a pair of homologous recombination sequences to be homologously recombined with the outside of the gene of interest in the linear genome-introduced nucleic acid fragment and a pair of endonuclease target sequences disposed on the side opposite to the position into which the linear genome-introduced nucleic acid fragment is incorporated via the homologous recombination sequences. ### Claim 3 (depends on 1) The method for producing a transformant according to claim 1, wherein the linear genome-introduced nucleic acid fragment comprises the pair of homologous recombination sequences to be incorporated into the predetermined position of the genome at positions sandwiching the gene of interest, the pair of endonuclease target sequences outside of the pair of homologous recombination sequences, and the pair of homologous recombination sequences for homologous recombination with the helper plasmid for transformation outside of the pair of endonuclease target sequences. ### Claim 4 (depends on 1) The method for producing a transformant according to claim 1, wherein the helper plasmid for transformation comprises a target-specific endonuclease gene that specifically cleaves the double strands of the endonuclease target sequences in an expressible state. ### Claim 5 (depends on 4) The method for producing a transformant according to claim 4, wherein the target-specific endonuclease gene is a homing endonuclease gene. ### Claim 6 (depends on 5) The method for producing a transformant according to claim 5, wherein the endonuclease target sequence is a sequence specifically recognized by homing endonuclease. ### Claim 7 (depends on 4) The method for producing a transformant according to claim 4, wherein the helper plasmid for transformation comprises an inducible promoter that regulates the expression of the target-specific endonuclease gene. ### Claim 8 (depends on 1) The method for producing a transformant according to claim 1, wherein the plurality of linear genome-introduced nucleic acid fragments consist of a first linear genome-introduced nucleic acid fragment to the nth linear genome-introduced nucleic acid fragment (wherein n is an integer of 2 or more), and the 3′-terminal side of the mth linear genome-introduced nucleic acid fragment (wherein m is an integer satisfying 1≤m≤n−1) has a sequence homologously recombined with the 5′-terminal side of the mth+1 linear genome-introduced nucleic acid fragment. ### Claim 9 A transformation method, comprising a step of introducing into a host, one or several types of linear genome-introduced nucleic acid fragments each comprising a gene of interest to be introduced into a predetermined position on a genome, and a helper plasmid for transformation having a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragments, wherein, in a state in which the linear genome-introduced nucleic acid fragments are incorporated into the helper plasmid for transformation, a pair of homologous recombination sequences for homologous recombination that takes place outside of the gene of interest and at the predetermined position on the genome, and a pair of endonuclease target sequences outside of the pair of homologous recombination sequences are disposed, wherein the gene of interest is expressed. ### Claim 10 (depends on 9) The transformation method according to claim 9, wherein the helper plasmid for transformation comprises a pair of homologous recombination sequences to be homologously recombined with the outside of the gene of interest in the linear genome-introduced nucleic acid fragment and a pair of endonuclease target sequences disposed on the side opposite to the position into which the linear genome-introduced nucleic acid fragment is incorporated via the homologous recombination sequences. ### Claim 11 (depends on 9) The transformation method according to claim 9, wherein the linear genome-introduced nucleic acid fragment comprises the pair of homologous recombination sequences to be incorporated into the predetermined position of the genome at positions sandwiching the gene of interest, the pair of endonuclease target sequences outside of the pair of homologous recombination sequences, and the pair of homologous recombination sequences for homologous recombination with the helper plasmid for transformation outside of the pair of endonuclease target sequences. ### Claim 12 (depends on 9) The transformation method according to claim 9, wherein the helper plasmid for transformation comprising a target-specific endonuclease gene that specifically cleaves the double strands of the endonuclease target sequences in an expressible state. ### Claim 13 (depends on 12) The transformation method according to claim 12, wherein the target-specific endonuclease gene is a homing endonuclease gene. ### Claim 14 (depends on 13) The transformation method according to claim 13, wherein the endonuclease target sequence is a sequence specifically recognized by homing endonuclease. ### Claim 15 (depends on 12) The transformation method according to claim 12, wherein the helper plasmid for transformation comprising an inducible promoter that regulates the expression of the target-specific endonuclease gene. ### Claim 16 (depends on 9) The transformation method according to claim 9, wherein the several types of linear genome-introduced nucleic acid fragments consist of a first linear genome-introduced nucleic acid fragment to the nth linear genome-introduced nucleic acid fragment (wherein n is an integer of 2 or more), and the 3′-terminal side of the mth linear genome-introduced nucleic acid fragment (wherein m is an integer satisfying 1≤m≤n−1) has a sequence homologously recombined with the 5′-terminal side of the mth+1 linear genome-introduced nucleic acid fragment. ### Claim 17 A helper plasmid for transformation, which is capable of incorporating into a genome, a linear genome-introduced nucleic acid fragment comprising a gene of interest to be introduced into a predetermined position on the genome, and which comprises a pair of homologous recombination sequences to be homologously recombined with the outside of the gene of interest in the linear genome-introduced nucleic acid fragment, and a pair of endonuclease target sequences disposed on the side opposite to the position into which the linear genome-introduced nucleic acid fragment is incorporated via the homologous recombination sequences. ### Claim 18 (depends on 17) The helper plasmid for transformation according to claim 17, comprising a target-specific endonuclease gene that specifically cleaves the double strands of the endonuclease target sequences in an expressible state. ### Claim 19 (depends on 18) The helper plasmid for transformation according to claim 18, wherein the target-specific endonuclease gene is a homing endonuclease gene. ### Claim 20 (depends on 19) The helper plasmid for transformation according to claim 19, wherein the endonuclease target sequence is a sequence specifically recognized by homing endonuclease. ### Claim 21 (depends on 18) The helper plasmid for transformation according to claim 18, comprising an inducible promoter regulating the expression of the target-specific endonuclease gene.