JP6190995B2 - 簡便で高効率の遺伝子改変非ヒト哺乳動物の作製方法 - Google Patents
簡便で高効率の遺伝子改変非ヒト哺乳動物の作製方法 Download PDFInfo
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Description
[1] 遺伝子改変された非ヒト哺乳動物の作製方法であって、Cas9タンパク質、標的DNA領域と相補的な塩基配列を含むcrRNA断片及びtracr RNA断片を非ヒト哺乳動物の卵母細胞に導入し、該標的DNAを遺伝子改変することを含む、上記方法。
[2] 非ヒト哺乳動物が、げっ歯類より選択される、[1]の方法。
[3] 卵母細胞が受精卵である、[1]又は[2]の方法。
[4] crRNA断片が、標的DNAと相補的な塩基配列、及び配列番号2で表される塩基配列もしくはその変異配列を含む、[1]〜[3]のいずれかの方法。
[5] tracr RNA断片が、配列番号4で表される塩基配列もしくはその変異配列を含む、[1]〜[4]のいずれかの方法。
[6] Cas9タンパク質、crRNA断片及びtracr RNA断片が複合体を形成している、[1]〜[5]のいずれかの方法。
[7] 遺伝子改変が標的DNA領域への遺伝子又は塩基配列の挿入であり、Cas9タンパク質、crRNA断片及びtracr RNA断片と共に、該遺伝子又は塩基配列を含むドナーDNAを非ヒト哺乳動物の卵母細胞に導入することを含む、[1]〜[6]のいずれかの方法。
[8] Cas9タンパク質1ng/μLに対し、crRNA断片及びtracr RNA断片をそれぞれ0.002pmol/μLを超える濃度で用いる、[1]〜[7]のいずれかの方法。
[9] 標的DNAと相補的な塩基配列、及び配列番号2で表される塩基配列もしくはその変異配列を含み、42塩基以下の塩基配列からなるcrRNA断片;ならびに/あるいは
配列番号4で表される塩基配列もしくはその変異配列を含み、69塩基以下の塩基配列からなるtracr RNA断片、
を含む標的DNAを遺伝子改変するためのキット。
[10] Cas9タンパク質、及び/又は、標的DNA領域へ挿入するための遺伝子もしくは塩基配列を含むドナーDNAをさらに含む、[9]のキット。
[11] 標的DNA領域へ遺伝子又は塩基配列が挿入されたマウスの作製方法であって、Cas9タンパク質、標的DNA領域と相補的な塩基配列を含むcrRNA断片及びtracr RNA断片、ならびに該遺伝子又は塩基配列を含むドナーDNAをマウスの卵母細胞に導入し、該標的DNA領域へ該遺伝子又は塩基配列を挿入することを含み、
ここで、該crRNA断片は30〜42塩基長であり、該tracr RNA断片は24〜69塩基長であり、該Cas9タンパク質を30ng/μL以上の濃度で、かつcrRNA断片及びtracr RNA断片をそれぞれ0.6pmol/μL以上の濃度で用いる、上記方法。
本発明においてCas9タンパク質は、CRISPR/Casシステムにおいて使用できるものであればよく、下記tracr RNA断片及びcrRNA断片と結合して活性化し、標的二本鎖DNAを切断できるものであればよく特に限定はされない。このようなCas9タンパク質は公知であり、WO2014/131833に例示されるものを利用することができる。好ましくは、Streptococcus pyogenes由来のCas9タンパク質を利用する。Cas9タンパク質のアミノ酸配列及び塩基配列は公開されたデータベース、例えば、GenBank(http://www.ncbi.nlm.nih.gov)に登録されており(例えば、アクセッション番号:Q99ZW2.1等)、本発明においてはこれらを利用することができる。
2.crRNA断片
本発明においてcrRNA断片は少なくとも、標的DNA領域と相補的な塩基配列とtracr RNA断片と相互作用可能な塩基配列を、5’側よりこの順で含んでなる。
3.tracr RNA断片
本発明においてtracr RNA断片は、5’側にcrRNA断片の一部の塩基配列と結合(ハイブリダイズ)可能な塩基配列を有し、これら塩基配列の相互作用によりcrRNA断片/tracr RNA断片ハイブリッドを形成する。これが標的DNA領域へのCas9タンパク質のガイドとして作用する。
4.ドナーDNA
本発明においてドナーDNAは、Cas9タンパク質に切断された部位にて生じる相同組換え修復(Homologous Recombination:HR)を利用して、標的DNA領域に所望の遺伝子又は塩基配列を挿入(ノックイン)するために用いられるDNAである。
5.遺伝子改変された非ヒト動物の作製方法
本発明の遺伝子改変された非ヒト哺乳動物の作製方法は、Cas9タンパク質、crRNA断片及びtracr RNA断片を非ヒト哺乳動物の卵母細胞に導入し、標的DNAを遺伝子改変することを含む。
(i) Cas9タンパク質は、5〜5000ng/μL、好ましくは5〜500ng/μL、より好ましくは10〜50ng/μL、さらに好ましくは20〜40ng/μL、よりさらに好ましくは30ng/μLとする;
(ii) crRNA断片及びtracr RNA断片はそれぞれ、Cas9タンパク質1ng/μLに対し、0.002pmol/μLを超える濃度、好ましくは0.005pmol/μL以上、より好ましくは0.01pmol/μL以上、さらに好ましくは0.02pmol/μL以上であり、上限は2pmol/μL以下、好ましくは0.2pmol/μL以下となる濃度とする(crRNA断片の量とtracr RNA断片の量とは、同一であってもよいし、異なっていてもよい);
(iii) crRNA断片及びtracr RNA断片はそれぞれ、0.06pmol/μLを超える濃度、好ましくは0.15pmol/μL以上、より好ましくは0.3pmol/μL以上、さらに好ましくは0.6pmol/μL以上であり、上限は60pmol/μL以下、好ましくは6pmol/μL以下、となる濃度とする(crRNA断片の量とtracr RNA断片の量とは、同一であってもよいし、異なっていてもよい)。
6.標的DNAを遺伝子改変するためのキット
本発明のキットは、crRNA断片及び/又はtracr RNA断片を含み、上記のとおり、標的DNAを遺伝子改変するため、ならびに/あるいは、標的DNAが遺伝子改変された非ヒト哺乳動物を作製するために用いることができる。
[実験材料及び方法]
(ターゲティングベクター)
ターゲティングベクターは、pAAV−TetO−FLEX−HA−mKate2−TeNT−polyAプラスミド(名古屋大学環境医学研究所神経系分野2 山中章弘博士より贈与)より以下のとおり作製した。まず、XhoI(NEB)及びHindIII(NEB)で消化してHA−mKate2−TeNTを除去し、そこにPCR増幅されたEGFPをコードする遺伝子を逆向きに置換・挿入した。次いで、NarI(NEB)及びBstEII(NEB)で消化してAAV2−ITRを除去し、そこにC57BL/6JマウスのゲノムDNAに由来するPCR増幅したβ−アクチン(以下、「Actb」と記載する)遺伝子の断片(2.0kb)を左ホモロジーアームとして、In−Fusion HD Cloning Kit(Takara)を用いて置換・挿入した。最後に、NotI(NEB)及びMluI(NEB)で消化し、そこにC57BL/6JマウスのゲノムDNAに由来するPCR増幅したActb遺伝子の断片(2.0kb)を右ホモロジーアームとしてIn−Fusion反応により置換・挿入した。
(Cas9タンパク質)
組換えCas9タンパク質は、NEB及びPNA Bio.より購入した。
(crRNA断片及びtracrRNA断片)
tracrRNA断片及びcrRNA断片は下記表1に示す塩基配列を有するものを化学合成し、ポリアクリルアミドゲル電気泳動により精製した(株式会社ファスマック)。crRNA断片はActb標的配列を含む。
1.濃度の検討
Cas9タンパク質(30ng/μL)とcrRNA断片及びtracrRNA断片(それぞれ、0pmol/μL,0.061pmol/μL,0.153pmol/μL,0.305pmol/μL、又は0.61pmol/μL)をActb標的配列を含むPCR産物と共に、Cas9 Nuclease Reactionバッファー(NEB)中にて37℃で60分間インキュベートし、次いでRNase A(5mg)で処理して(37℃にて30分間)、RNAを除去した。反応を30%グリセロール、1.2%SDS及び250mM EDTAを含む6×DNAローディングバッファーで停止し、反応物を2%アガロースゲルにて電気泳動した。対照には、crRNA断片、及びtracrRNA断片を加えなかった。
2.crRNA断片長の検討
上記1.の実験においてcrRNA断片を、下記表2に示す塩基配列を有するものに代え、それぞれ0.61pmol/μLにて用いた以外は同一の条件にてin vitro切断アッセイを行った。対照には、いずれのcrRNA断片も加えなかった。
0.1TEバッファー中に、Cas9タンパク質(30ng/μL)、crRNA断片(0.061もしくは0.61pmol/μL)、tracrRNA断片(0.061もしくは0.61pmol/μL)、及びpActb−TetO−FLEX−EGFP−polyA(10ng/μL)を添加・混合し、37℃にて少なくとも15分間インキュベートし、複合体を形成した。
(PCRスクリーニング)
産仔の尾部を一部採取し、プロテイナーゼKで処理した後、フェノール抽出法によりゲノムDNAを調製した。次いで、得られたゲノムDNAを鋳型として、ExTaq(Takara)及び下記表3に示す3種のプライマー対を用いてPCRを行い、1%アガロースゲルにて電気泳動してノックインマウスをスクリーニングした。得られたPCR産物についてはさらに、TOPO TA Cloning Kit(Life Technologies)を用いてクローニングし、配列決定を行った。
サザンプローブ(0.8kb)は、BDF1ゲノムDNAよりPCR増幅し、TOPO TA Cloning Kitを用いてクローニングした後、32Pランダムプライマー(Perkin Elmer)を用いて標識して調製した。ノックインマウスより得たゲノムDNAは、EcoRIで消化して0.8%アガロースゲルを用いた電気泳動により分離し、ナイロンメンブレン(Amersham)へ転写した後、サザンプローブとハイブリダイズさせ検出し、遺伝子型を確認した。プローブの位置は図2に示す。
(繊維芽細胞の初代培養)
2週齢のマウスの耳より小片を切り取り、37℃にて30分間、4mg/mlコラゲナーゼL(新田ゼラチン株式会社)及び4mg/mlディスパーゼで処理した後、10%FBS/DMEM中、37℃、10%CO2条件下にて数日間培養した。pCAG−Cre、pCMV−tTA(Takara)及びpCMV−DsRed(Takara)をLipofectamine(登録商標)LTX & Plus reagent(Life Technologies)を用いて細胞にコトランスフェクトし、EGFPの発現を蛍光顕微鏡にて確認した。
[結果]
(in vitro切断アッセイ)
1.濃度の検討結果
結果を図3−1に示す。
2.crRNA断片長の検討結果
結果を図3−2に示す。
(ノックインマウスの作製)
CRISPR/CASシステムにて一般的に用いられるRNA量「0.061pmol/μL」にてcrRNA断片及びtracrRNA断片を、Cas9タンパク質及びpActb−TetO−FLEX−EGFP−polyAと共に受精卵の前核に注入した結果、9匹の産仔を得ることができた。得られた産仔についてPCRスクリーニングしたところ、Actb遺伝子座にTetO−FLEX−EGFP−polyAカセットを保有するマウスは確認できなかった(下記表4)。
得られたノックインマウス及び野生型マウスの耳介より採取した繊維芽細胞をそれぞれ培養し、これにpCAG−Cre、pCMV−tTA及びpCMV−DsRedをコトランスフェクトしたところ、ノックインマウスに由来する繊維芽細胞においてEGFPの蛍光が観察された(図5:矢頭)。特に、ノックインアレルをホモ接合に有するマウスにおいて強いシグナルが確認された。
Claims (6)
- 標的DNA領域へ遺伝子又は塩基配列が挿入されたマウスの作製方法であって、Cas9タンパク質、標的DNA領域と相補的な塩基配列を含むcrRNA断片及びtracr RNA断片、ならびに該遺伝子又は塩基配列を含むドナーDNAをマウスの卵母細胞に導入し、該標的DNA領域へ該遺伝子又は塩基配列を挿入することを含み、
ここで、該crRNA断片は30〜42塩基長であり、該tracr RNA断片は24〜69塩基長であり、該Cas9タンパク質を30〜500ng/μLの濃度で、かつcrRNA断片及びtracr RNA断片をそれぞれ0.6pmol/μL以上の濃度で用いる、上記方法。 - 卵母細胞が受精卵である、請求項1に記載の方法。
- crRNA断片が、標的DNAと相補的な塩基配列、及び配列番号2で表される塩基配列もしくはそれと90%以上の同一性を有する塩基配列を含む、請求項1又は2に記載の方法。
- tracr RNA断片が、配列番号4で表される塩基配列もしくはそれと90%以上の同一性を有する塩基配列を含む、請求項1〜3のいずれか1項に記載の方法。
- Cas9タンパク質、crRNA断片及びtracr RNA断片が複合体を形成している、請求項1〜4のいずれか1項に記載の方法。
- Cas9タンパク質1ng/μLに対し、crRNA断片及びtracr RNA断片をそれぞれ0.002pmol/μLを超える濃度で用いる、請求項1〜5のいずれか1項に記載の方法。
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WO2014131833A1 (en) * | 2013-02-27 | 2014-09-04 | Helmholtz Zentrum München Deutsches Forschungszentrum Für Gesundheit Und Umwelt (Gmbh) | Gene editing in the oocyte by cas9 nucleases |
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