JP2020031546A - ゲノム編集技術 - Google Patents
ゲノム編集技術 Download PDFInfo
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- JP2020031546A JP2020031546A JP2018158921A JP2018158921A JP2020031546A JP 2020031546 A JP2020031546 A JP 2020031546A JP 2018158921 A JP2018158921 A JP 2018158921A JP 2018158921 A JP2018158921 A JP 2018158921A JP 2020031546 A JP2020031546 A JP 2020031546A
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Abstract
Description
本発明は、その一態様において、ゲノム編集用ヌクレアーゼのコード配列、及び該コード配列の3’側に配置されたnanos 3遺伝子3’UTRを含む、ポリヌクレオチド(本明細書において、「本発明のポリヌクレオチド」と示すこともある。)に関する。以下、これについて説明する。
nanos 3遺伝子3’UTRは、その活性を損なわない限りにおいて、塩基配列の変異(例えば、置換、欠失、挿入、付加等)を有していてもよい。この観点から、nanos 3遺伝子3’UTRは、野生型nanos 3遺伝子3’UTRの塩基配列と、例えば85%以上、好ましくは90%以上、より好ましくは95%以上、さらに好ましくは98%以上の同一性を有する塩基配列であってもよい。或いは、同様の観点から、nanos 3遺伝子3’UTRは、野生型nanos 3遺伝子3’UTRの塩基配列に対して1若しくは複数個(例えば2〜8個、好ましくは2〜5個、より好ましくは2〜3個、さらに好ましくは2個)の塩基が置換、欠失、付加、又は挿入された塩基配列であってもよい。
本発明のポリヌクレオチドは、ゲノム編集用組成物として利用することもできるし、或いはゲノム編集用キットとして利用することもできる。
本発明のポリヌクレオチドを生物又は細胞に導入する工程を含む方法により、これらの生物又は細胞のゲノムを編集することができる。
まず、nanos 3遺伝子3’UTRを含むCas9 mRNAの発現ベクター(pCS2+hSpCas9-nos3 3’UTR (Cas9-nos3UTR))を調製した。該ベクターの調製方法の概略を図1に示す。図1中に示される塩基配列の内、「Cas9-nos3UTR」の塩基配列は配列番号1に示し、「nos3UTR」の塩基配列は配列番号2に示す。nanos3 cDNAを鋳型として、5’側にXbaI認識配列が付加されたプライマーセット(nos3-F-XbaI:配列番号3と、nos3-R-XbaI:配列番号4とのセット)を用いてPCRを行い、nanos 3遺伝子3’UTRを含むDNA断片を得た。得られたDNA断片をXbaIで消化し、pCS2+hSpCas9 (addgene #51815)のXbaIサイトへ挿入して、pCS2+hSpCas9-nos3 3’UTR (Cas9-nos3UTR)を得た。
<実施例2-1.卵細胞へのベクターの注入、及び生育>
ゲノム編集対象として、生殖細胞をEGFPで可視化できるolvas-EGFPと軟骨細胞及び生殖腺体細胞をDsRedで可視化できるsox9b-DsRedの二重トランスジェニックメダカ(PNAS, vol.98, No.5, 2544-2549, 2001.)の受精卵を用いた。Cas9-nos3 3’UTR mRNA又はCas9 mRNA(濃度50ng/μl-100ng/μl )と、EGFP及びDsRed遺伝子をターゲットとするgRNA(濃度10ng/μl-50ng/μl)とを、ガラスキャピラリー(Warner Instruments, G100F-4)に充填し、マイクロインジェクター FemtoJet (eppendorf)を用いて、メダカ受精卵の1細胞期に注入した。注入後、得られた受精卵を発生させ、受精後7日のメダカ胚を得た。
実施例蛍光実体顕微鏡SZX12(オリンパス)を用いて、メダカ胚のEGFP及びDsRedの蛍光を観察し、顕微鏡用カメラDP74(オリンパス)で撮影した。
観察像の例を図2に示す。生殖細胞が1つでも残って光っているメダカ胚をEGFR(+)と判定し、生殖細胞が全く光っていないメダカ胚、すなわちすべての生殖細胞のEGFP 遺伝子が破壊されているメダカ胚をEGFR(-)と判定した。
Claims (15)
- ゲノム編集用ヌクレアーゼのコード配列、及び該コード配列の3’側に配置されたnanos 3遺伝子3’UTRを含む、ポリヌクレオチド。
- 前記ゲノム編集用ヌクレアーゼがCasタンパク質である、請求項1に記載のポリヌクレオチド。
- 前記nanos 3遺伝子が動物由来nanos 3遺伝子である、請求項1又は2に記載のポリヌクレオチド。
- 前記ゲノム編集用ヌクレアーゼが、核移行シグナルが付加されたゲノム編集用ヌクレアーゼである、請求項1〜3のいずれかに記載のポリヌクレオチド。
- mRNAである、請求項1又は2に記載のポリヌクレオチド。
- ベクターである、請求項1〜4のいずれかに記載のポリヌクレオチド。
- 前記ゲノム編集用ヌクレアーゼのコード配列の5’側に配置されたプロモーターを含む、請求項6に記載のポリヌクレオチド。
- さらに、ガイドRNA発現カセットを含む、請求項6又は7に記載のポリヌクレオチド。
- 請求項1〜8のいずれかに記載のポリヌクレオチドを含む、細胞。
- 請求項1〜8のいずれかに記載のポリヌクレオチドを含む、ゲノム編集用組成物。
- 請求項1〜8のいずれかに記載のポリヌクレオチドを含む、ゲノム編集用キット。
- 請求項1〜8のいずれかに記載のポリヌクレオチドを生物又は細胞に導入する工程を含む、ゲノム編集方法。
- 前記ポリヌクレオチドの導入対象が受精卵である、請求項12に記載のゲノム編集方法。
- 請求項1〜8のいずれかに記載のポリヌクレオチドを生物又は細胞に導入する工程を含む、ゲノム編集された生物又は細胞の製造方法。
- 前記ポリヌクレオチドの導入対象が受精卵である、請求項14に記載の製造方法。
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