内容の要約広島大学(Hiroshima University)博士(理学)Doctor of Sciencedoctora

Nakamae, Kazuki

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内容の要約博士(理学)Doctor of Science広島大

Hiroshima University Institutional Repository

ㄽ ᩥ ᑂ ᰝ ࡢ せ ᪨ ༤ኈࡢᑓᨷศ㔝ࡢྡ⛠  ༤ ኈ㸦 ⌮Ꮫ 㸧 Ặྡ ୰๓ ࿴ᜤ Ꮫ఩ᤵ୚ࡢせ௳ Ꮫ఩つ๎➨ 4᮲➨ 1࣭2㡯ヱᙜ ㄽᩥ㢟┠ Automated design and detailed profiling of MMEJ-assisted knock-in using a newly constructed computational pipeline 㸦᪂つ䛻ᵓ⠏䛧䛯䝁䞁䝢䝳䞊䝍䝟䜲䝥䝷䜲䞁䜢฼⏝䛧䛯 㻹㻹㻱㻶 䝜䝑䜽䜲䞁䛾⮬ືタィཬ䜃ヲ⣽䝥䝻䝣䜯䜲䝸䞁䜾㸧 ㄽᩥᑂᰝᢸᙜ⪅ ୺  ᰝ   ᩍ ᤵ  ᒣᮏ ༟ ᑂᰝጤဨ   ᩍ ᤵ  ஭ฟ ༤                  ᑂᰝጤဨ   ᩍ ᤵ  ᆏᮏ ᩔ                  ᑂᰝጤဨ   ㅮ ᖌ  బஂ㛫 ဴྐ                ࠝㄽᩥᑂᰝࡢせ᪨ࠞ  ࢤࣀ࣒⦅㞟ࡣࠊேᕤ DNAษ᩿㓝⣲ࢆ⏝࠸࡚ DNA஧ᮏ㙐ษ᩿ (DSB)ࢆᑟධࡋࠊࡑࡢಟ᚟㐣⛬࡛ᶆⓗ㑇ఏᏊࡢ◚ቯ (㑇ఏᏊࣀࢵࢡ࢔࢘ࢺ)ࡸእ᮶㑇ఏᏊࡢᤄධ (㑇ఏᏊࣀࢵࢡ࢖ࣥ)ࢆ⾜࠺ᢏ⾡࡛࠶ࡿࠋ㏆ᖺࠊேᕤ DNAษ᩿㓝⣲ࡢ CRISPR-Cas9ࢆ⏝࠸ࡓ࣐࢖ࢡࣟ࣍ࣔࣟࢪ࣮፹௓ᮎ➃⤖ྜ㸦MMEJ㸧࡟ࡼࡿຠ⋡ⓗ࡞㑇ఏᏊࣀࢵࢡ࢖ࣥᢏ⾡࡜ࡋ࡚ PITChἲ࠾ࡼࡧ LoADἲࡀ㛤Ⓨࡉࢀᵝࠊ ࠎ࡞ᇵ㣴⣽⬊ᰴࡸື≀✀࡛ࡑࡢ᭷ຠᛶࡀ♧ࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊPITChἲࡸ LoADἲ࡟฼⏝ࡍࡿᶆⓗ DNAࡢ≉ᚩࡸࢻࢼ࣮DNAࡢࢹࢨ࢖ࣥ࡟ࡘ࠸࡚ࠊᐇ㦂ゎᯒࢹ࣮ࢱࢆ⤌ࡳྜࢃࡏࡓ᭱㐺໬◊✲ࡣ㐍ࢇ࡛࠸࡞࠸ࠋࡑࡇ࡛ᮏㄽᩥࡢⴭ⪅ࡣࠊMMEJ ࢆ௓ࡋࡓࣀࢵࢡ࢖ࣥ࡟㛵ࢃࡿせᅉࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ࡟ࠊ㓄ิタィ࠿ࡽゎᯒࡲ࡛ࢆ⏬୍ⓗ࡟ᐇ᪋ࡍࡿࢯࣇࢺ࢙࢘࢔ࣃ࢖ࣉࣛ࢖ࣥࢆ㛤Ⓨࡋࡓࠋࡉࡽ࡟ࠊ40 㑇ఏᏊᗙ࡬ࡢ㑇ఏᏊࣀࢵࢡ࢖ࣥᐇ㦂ࢆ⾜࠸ࠊࡑࡢḟୡ௦ࢩ࣮ࢣࣥࢫ(NGS)ࢹ࣮ࢱ࠿ࡽ MMEJ ࣀࢵࢡ࢖ࣥࡢຠ⋡࡟୚࠼ࡿ✀ࠎࡢᅉᏊࡢᙳ㡪ࢆゎᯒࡋࡓࠋ  ➨୍❶࡛ࡣࠊ᭱㐺࡞㑇ఏᏊࣀࢵࢡ࢖ࣥࢆ⾜࠺ࡓࡵࠊMMEJ ࣀࢵࢡ࢖ࣥࡢ㓄ิࢹࢨ࢖ࣥἲࡢᐃ⩏ࢆ⾜࡞ࡗࡓࠋࡑࡢᐃ⩏ࡢࡓࡵࡢ࢔ࣉ࣮ࣟࢳ࡜ࡋ࡚ MMEJࣀࢵࢡ࢖ࣥࢆ⏬୍ⓗ࡟⮬ືタィࡍࡿࢯࣇࢺ࢙࢘࢔͆PITCh designer͇ࢆ JavaScript+HTML5 ࢆ⏝࠸࡚㛤ⓎࡋࡓࠋPITCh designer ࡣ௵ពࡢᶆⓗ㓄ิࡢ୰࠿ࡽ MMEJ ࣀࢵࢡ࢖ࣥࡢࢹࢨ࢖ࣥࡀྍ⬟࡞㡿ᇦࢆ᳨⣴ࡋࠊ୍ᮏ㙐࢞࢖ࢻ RNA㸦sgRNA㸧ࡸ࣐࢖ࢡࣟ࣍ࣔࣟࢪ࣮㓄ิ࠾ࡼࡧࢻࢼ࣮ᵓ⠏࡟ᚲせ࡞ࣉࣛ࢖࣐࣮᝟ሗ࡞࡝ࢆᥦ౪ࡍࡿࠋࡇࢀ࡟ࡼࡾᇶᮏⓗ࡞MMEJࣀࢵࢡ࢖ࣥࡢࢹࢨ࢖ࣥࢆࢥࣥࣆ࣮ࣗࢱ࢔ࣝࢦࣜࢬ࣒ࡢ࡛ࣞ࣋ࣝᐃ⩏ࡍࡿࡇ࡜࡟ᡂຌࡋࡓࠋຍ࠼࡚ࠊᵝࠎ࡞ࢱ࢖ࣉࡢ CRISPR-Cas9 ࡛ࡢ MMEJ ࣀࢵࢡ࢖ࣥࢆྍ⬟࡜ࡍࡿ Web ࢶ࣮ࣝ࡜ࡋ࡚බ㛤ࡍࡿࡇ࡜࡟ࡼࡾࠊࣀࢵࢡ࢖ࣥࡢࢹࢨ࢖ࣥࡢ฼౽ᛶࢆྥୖࡋࡓࠋ  ➨஧❶࡛ࡣࠊ➹⪅ࡣ MMEJ ࣀࢵࢡ࢖ࣥࡢഴྥࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࡢ NGS ゎᯒࣃ࢖ࣉࣛ࢖ࣥ࡜ࡋ࡚͆MMEJ-assisted chromosomal integration analysis tools (MaChIAto)”ࢆ㛤ⓎࡋࡓࠋMaChIAto ࡣࠊ᪤Ꮡࡢኚ␗ゎᯒỗ⏝ࢶ࣮ࣝ CRISPResso ࡜㐃ᦠࡋ࡚ࡼࡾໟᣓⓗ࡞ኚ␗ゎᯒ࠾ࡼࡧᩘⓒ✀࡟ࢃࡓࡿ≉ᚩゎᯒࢆྍ⬟࡜ࡋࡓࠋࡲࡓኚ␗ศ㢮ࢆࡼࡾṇ☜࡞ࡶࡢ࡬࡜෌ศ㢮ࡍࡿᶵ⬟ࢆᦚ㍕ࡋࡓࠋேᕤⓗ࡞㓄ิࢆ⏝࠸࡚ゎᯒࢆ⾜ࡗࡓ࡜ࡇࢁࠊࡑࡢᛶ⬟ࡣᚑ᮶ࡢኚ␗ศ㢮ゎᯒࢶ࣮ࣝࡢ୰࡛ࡶࢺࢵࣉࢡࣛࢫࡢṇ☜ᛶࢆ♧ࡍࡇ࡜ࡀ☜ㄆࡉࢀࡓࠋMaChIAto ࡢᶵ⬟ᛶࢆ᳨ドࡍࡿࡓࡵࠊPITCh designer ࡛タィࡋࡓ 40 ✀㢮ࡢ㑇ఏᏊᗙ࡟ᑐࡍࡿ MMEJ ࣀࢵࢡ࢖ࣥࢹ࣮ࢱࢆ MaChIAto ࡟ࡼࡾゎᯒࡋࡓࠋࡑࡢ⤖ᯝࠊ40 㑇ఏᏊᗙࡢ඲࡚࡟࠾࠸࡚ PITChἲ࠾ࡼࡧ LoADἲ࡟ࡼࡿṇ☜࡞ࣀࢵࢡ࢖ࣥ NGSࢹ࣮ࢱࢆᚓࡿࡇ࡜ࡀ࡛ࡁࡓࠋࡲࡓ PITCh ἲ࡛ࡢࣀࢵࢡ࢖ࣥࡢṇ☜ᛶࡣࠊCRISPR-Cas9 ࡟ࡼࡿ࢖ࣥࢹࣝᙧᡂ⬟ຊ࡟౫Ꮡࡏࡎࠊࢻࢼ࣮ࡀࡶࡘ࣐࢖ࢡࣟ࣍ࣔࣟࢪ࣮㓄ิࡢ⇕ຊᏛⓗ≉ᛶࠊ஧ḟᵓ㐀ᅉᏊࠊ࠶ࡿ࠸ࡣ㓄ิࢥࣥࢸ࢟ࢫࢺࡸᶆⓗ࿘㎶࡬ࡢ࢔ࢡࢭࢫᛶ࡟౫Ꮡࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡉࡽ࡟ࠊLoADἲ࡛ࡢࣀࢵࢡ࢖࡛ࣥࡣ㢮ఝࡋࡓഴྥࢆ♧ࡍ୍᪉࡛ࠊPITChἲ࡛ࡢࣀࢵࢡ࢖ࣥࡼࡾࡶࡇࢀࡽᅉᏊ࡟ᑐࡍࡿ౫Ꮡᛶࡀ⦆࿴ࡉࢀ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋḟ࡟ࠊMMEJ ࣀࢵࢡ࢖ࣥ࡜ྠ᫬࡟ぢࡽࢀࡿ࢖ࣥࢹࣝࣃࢱ࣮ࣥ࡜ࣀࢵࢡ࢖ࣥຠ⋡࡟╔┠ࡋࡓゎᯒࢆ⾜ࡗࡓࠋ㧗ຠ⋡࡞ࣀࢵࢡ࢖ࣥࢆ♧ࡋࡓ㑇ఏᏊᗙࢢ࣮ࣝࣉ࡛ࡣ 3ሷᇶ௨ୖࡢḞኻࡀከࡃぢࡽࢀࡿ࡟ᑐࡋࠊపຠ⋡࡞ࣀࢵࢡ࢖ࣥࢆ♧ࡋࡓ㑇ఏᏊᗙࢢ࣮ࣝࣉ࡛ࡣ㸯ሷᇶᤄධࡀ㧗㢖ᗘ࡟ほᐹࡉࢀࡓࠋࡇࢀ࡟㛵ࡋ࡚㓄ิⓗ࡞≉ᚩ࡟ὀ┠ࡋࡓ࡜ࡇࢁࠊ㧗ຠ⋡࡞ࣀࢵࢡ࢖ࣥࢆ♧ࡋࡓ㑇ఏᏊᗙࢢ࣮ࣝࣉࡣGࣜࢵࢳ࡞࣐࢖ࢡࣟ࣍ࣔࣟࢪ࣮㓄ิࢆ௓ࡋࡓMMEJࡀẚ㍑ⓗከࡃほᐹࡉࢀࠊࡉࡽ࡟ 3ሷᇶ࿘ᮇ㸦6, 9, 12 bp➼㸧ࡢḞኻࡀ㢧ⴭ࡟☜ㄆࡉࢀࡓࠋ୍᪉࡛పຠ⋡࡞ࣀࢵࢡ࢖ࣥࢆ♧ࡋࡓ㑇ఏᏊᗙࢢ࣮ࣝࣉࡢ୰ࡢ࠸ࡃࡘ࠿ࡢ㑇ఏᏊᗙ࡛ࡣࠊCRISPR-Cas9࡟ࡼࡿษ᩿㠃ࡢ T ሷᇶࡢࡼ࠺࡞୍ሷᇶㄏᑟᛶࢆࡶࡘ㓄ิ≉ᚩࢆ᭷ࡋ࡚࠸ࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊMMEJ ࣀࢵࢡ࢖ࣥࡢຠ⋡ࡣ࣐࢖ࢡࣟ࣍ࣔࣟࢪ࣮ෆࡢ㓄ิᏑᅾẚࡢࡳ࡞ࡽࡎሷᇶ㓄ิࡢ㓄⨨࡟ࡶ౫Ꮡࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ௨ୖ➨஧❶ࡢ⤖ᯝ࠿ࡽࠊ➹⪅ࡢ㛤ⓎࡋࡓMaChIAtoࡢ฼⏝࡟ࡼࡾMMEJࣀࢵࢡ࢖ࣥࡢഴྥࡢ⌮ゎ࡟ࡘ࡞ࡀࡿᵝࠎ࡞≉ᚩࢆ᥎ ࡛ࡁࡿྍ⬟ᛶࡀ♧ࡉࢀࡓࠋ  ௨ୖᮏ◊✲࡛ࡣࠊࢤࣀ࣒⦅㞟ᢏ⾡࡜ࡋ࡚ỗ⏝ⓗ࡞ MMEJࣀࢵࢡ࢖ࣥࡢࢹࢨ࢖ࣥ࠿ࡽゎᯒ࡟⮳ࡿࡲ࡛ࡢ୍㐃ࡢࢯࣇࢺ࢙࢘࢔ࣃ࢖ࣉࣛ࢖ࣥࢆ㛤Ⓨࡋࡓࠋࡇࢀࡽࡢࢶ࣮ࣝࢆ฼⏝ࡍࡿࡇ࡜࡛ PITChἲ࡜ LoADἲ࡟ࡼࡿ㑇ఏᏊࣀࢵࢡ࢖ࣥࡢഴྥࢆゎᯒ࡛ࡁࡿࡇ࡜ࢆド᫂ࡋࠊ㸦࢚ࣆ㸧ࢤࣀ࣒ࢥࣥࢸ࢟ࢫࢺ࡜࢖ࣥࢹࣝࣃࢱ࣮ࣥࡀࣀࢵࢡ࢖ࣥࡢຠᯝ࡟ᐤ୚ࡋ࡚࠸ࡿࡇ࡜ࢆ♧၀ࡋࡓࠋᮏ◊✲ࡣMMEJࣀࢵࢡ࢖ࣥࡢഴྥࢆึࡵ࡚グ㏙ࡋࡓᇶ┙ⓗ࡞ᡂᯝ࡛࠶ࡿࡔࡅ࡛࡞ࡃࠊࡼࡾ᝟ሗ⛉Ꮫⓗ࡞どⅬ࡛ࢤࣀ࣒ᕤᏛᢏ⾡㛤Ⓨࢆ㐍ࡵࡿࡓࡵࡢࣉࣛࢵࢺࣇ࢛࣮࣒ᙧᡂ࡟ࡶᐤ୚ࡋࡓ࡜⪃࠼ࡽࢀࡿࠋ  ௨ୖ㸪ᑂᰝࡢ⤖ᯝ㸪ᮏㄽᩥࡢⴭ⪅ࡣ༤ኈ㸦⌮Ꮫ㸧ࡢᏛ఩ࢆᤵ୚ࡉࢀࡿ༑ศ࡞㈨᱁ࡀ࠶ࡿࡶࡢ࡜ㄆࡵࡿࠋ          බ⾲ㄽᩥ Establishment of expanded and streamlined pipeline of PITCh knock-in - a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO. Kazuki Nakamae, Yuki Nishimura, Mitsumasa Takenaga, Shota Nakade, Naoaki Sakamoto, Hiroshi Ide, Tetsushi Sakuma and Takashi Yamamoto. Bioengineered, 8, 302-308 (2017)  ཧ⪃ㄽᩥ Biased genome editing using the local accumulation of DSB repair molecules system. Shota Nakade, Keiji Mochida, Atsushi Kunii, Kazuki Nakamae, Tomomi Aida, Kohichi Tanaka, Naoaki Sakamoto, Tetsushi Sakuma and Takashi Yamamoto. Nature Communications, 9, 3270 (2018)                           

Automated design and detailed profiling of MMEJ-assisted knock-in using a newly constructed computational pipeline

http://ir.lib.hiroshima-u.ac.jp/files/public/4/49452/20200721120052878153/k8155_2.pdf

新規に構築したコンピュータパイプラインを利用したMMEJノックインの自動設計及び詳細プロファイリング

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