CRISPR-BASED GENOME MODIFICATION AND REGULATION

• Main Authors: GREGORY D DAVIS, KANG QIAOHUA, CHEN FUQIANG, SCOTT W KNIGHT
• Format: Patent
• Language: English; Japanese
• Published: 26.10.2017
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

PROBLEM TO BE SOLVED: To provide a method for modifying a chromosomal sequence in an eukaryotic cell.SOLUTION: According to the present invention, there is provided a method for modifying a chromosomal sequence by incorporating a donor sequence, the method comprising: (a) introducing, into an eukaryotic cell, (i) a RNA-guided endonuclease comprising a nuclear localization signal or a codon-optimized nucleic acid encoding the RNA-guided endonuclease comprising the nuclear localization signal, (ii) a guide RNA or DNA encoding the guide RNA, and (iii) a donor polynucleotide comprising a donor sequence; and (b) culturing the eukaryotic cell such that the guide RNA guides the RNA-guided endonuclease to a target site in a chromosomal sequence where the RNA-guided endonuclease induces cleavage of a double strand at the target site, and the cleavage of the double strand is modified by a DNA repair process to modify the chromosomal sequence by insertion or substitution of a donor sequence into the chromosomal sequence.SELECTED DRAWING: None 【課題】真核細胞において染色体配列を修飾するための方法の提供。【解決手段】ａ）真核細胞に、（ｉ）核局在化シグナルを含むＲＮＡ誘導型エンドヌクレアーゼ、又は核局在化シグナルを含むＲＮＡ誘導型エンドヌクレアーゼをコードするコドン最適化された核酸、（ｉｉ）ガイドＲＮＡ又はガイドＲＮＡをコードするＤＮＡ、及び（ｉｉｉ）ドナー配列を含むドナーポリヌクレオチドを導入すること、及びｂ）真核細胞を、ガイドＲＮＡが、ＲＮＡ誘導型エンドヌクレアーゼを染色体配列中の標的部位へ誘導し、そこでＲＮＡ誘導型エンドヌクレアーゼが、標的部位にて二本鎖の切断を誘導し、二本鎖の切断が、染色体配列が、ドナー配列の染色体配列への挿入又は置換により修飾されるようにＤＮＡ修復過程により修復されるように培養すること、を含む、ドナー配列を組み込むことによる、染色体配列を修飾するための方法。【選択図】なし