JP6620018B2 - Crisprに基づくゲノム修飾および制御 - Google Patents
Crisprに基づくゲノム修飾および制御 Download PDFInfo
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Description
本発明は、標的ゲノム修飾に関する。特に、本発明は、RNA誘導型エンドヌクレアーゼまたはCRISPR/Cas様タンパク質を含む融合タンパク質、および標的染色体配列を修飾または調節するための該タンパク質の使用方法に関する。
標的ゲノム修飾は、真核細胞、胚、および動物の遺伝子操作のための強力なツールである。例えば、外来性配列は、標的ゲノム部位に組み込まれ、および/または特定の内在性染色体配列は、欠失、不活性化または修飾され得る。現在の方法は、例えば、ジンクフィンガーヌクレアーゼ(ZFN)または転写アクティベーター様エフェクターヌクレアーゼ(TALEN)のような人工ヌクレアーゼの使用に依存している。これらのキメラヌクレアーゼは、非特異的DNA切断ドメインに連結したプログラム可能な配列特異的DNA結合分子を含む。しかしながら、それぞれの新規ゲノム標的は、新規の配列特異的DNA結合分子を含む新規ZFNまたはTALENの設計を必要とする。従って、これらのカスタム設計されたヌクレアーゼは、割高な費用が掛かり、製造に時間を要する傾向がある。さらに、ZFNおよびTALENの特異性は、それらがオフターゲット(off−target)の切断を仲介し得る程度のものである。
本開示の種々の面には、単離されたRNA誘導型エンドヌクレアーゼの提供であって、該エンドヌクレアーゼが、少なくとも1つの核局在化シグナル、少なくとも1つのヌクレアーゼドメイン、および切断のために特定のヌクレオチド配列にエンドヌクレアーゼを標的化するガイドRNAと相互作用する少なくとも1つのドメインを含む、エンドヌクレアーゼの提供が含まれる。一態様において、エンドヌクレアーゼは、Cas9タンパク質に由来し得る。別の態様において、エンドヌクレアーゼは、少なくとも1つの機能的ヌクレアーゼドメインを欠失するように修飾され得る。他の態様において、エンドヌクレアーゼは、細胞透過性ドメイン、マーカードメイン、または両方をさらに含み得る。別の態様において、エンドヌクレアーゼは、ガイドRNAを含むタンパク質−RNA複合体の一部であり得る。いくつかの例において、ガイドRNAは、標的部位に相補的な5’領域を含む単一分子であり得る。また、本明細書に記載のRNA誘導型エンドヌクレアーゼの何れかをコードする単離された核酸も提供する。いくつかの態様において、核酸は、例えばヒト細胞のような哺乳動物細胞における翻訳に最適化されているコドンであり得る。他の態様において、RNA誘導型エンドヌクレアーゼをコードする核酸配列は、プロモーター調節配列に操作可能に連結されていてよく、要すれば、ベクターの一部であってよい。他の態様において、プロモーター調節配列に操作可能に連結されていてよいRNA誘導型エンドヌクレアーゼをコードする配列を含むベクターはまた、プロモーター調節配列に操作可能に連結されていてよいガイドRNAをコードする配列も含み得る。
本発明は、少なくとも1つの核局在化シグナル、少なくとも1つのヌクレアーゼドメイン、および切断のためにエンドヌクレアーゼを特定のヌクレオチド配列に標的化するガイドRNAと相互作用する少なくとも1つのドメインを含む、RNA誘導型エンドヌクレアーゼを提供する。また、RNA誘導型エンドヌクレアーゼをコードする核酸、ならびに真核細胞または胚の染色体配列を修飾するための該RNA誘導型エンドヌクレアーゼの使用方法を提供する。RNA誘導型エンドヌクレアーゼは、それぞれがエンドヌクレアーゼを特定の標的部位に誘導し、その部位で、RNA誘導型エンドヌクレアーゼが、染色体配列が修飾されるようにDNA修復過程により修復され得る二本鎖の切断を導入する、複数の特定のガイドRNAと相互作用する。特異性がガイドRNAにより提供されるため、RNAベースのエンドヌクレアーゼが汎用され、異なるガイドRNAを用いて異なるゲノム配列を標的とするために使用され得る。本明細書に記載の方法は、特定の染色体配列を標的とし、修飾するため、および/または細胞または胚のゲノムにおける標的領域に内在性配列を導入するために使用され得る。さらに、標的化は、特異的であり、オフターゲット作用は制限されている。
本明細書に記載の一面は、真核細胞および胚、例えば、非ヒト単一細胞胚などの核へのエンドヌクレアーゼの移入を可能にする、少なくとも1つの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提供する。RNA誘導型エンドヌクレアーゼはまた、少なくとも1つのヌクレアーゼドメインおよびガイドRNAと相互作用する少なくとも1つのドメインを含む。RNA誘導型エンドヌクレアーゼは、ガイドRNAによって特定の核酸配列(または標的部位)に誘導される。ガイドRNAは、一旦標的部位に誘導されると、RNA誘導型エンドヌクレアーゼが標的部位の核酸配列に二本鎖の切断を導入し得るように、RNA誘導型エンドヌクレアーゼならびに標的部位を相互作用させる。ガイドRNAは標的化切断のための特異性を提供するため、RNA誘導型エンドヌクレアーゼのエンドヌクレアーゼは汎用性であり、異なる標的核酸配列を切断するために異なるガイドRNAと共に用いることができる。本発明は、単離されたRNA誘導型エンドヌクレアーゼ、RNA誘導型エンドヌクレアーゼをコードする単離された核酸(すなわち、RNAまたはDNA)、RNA誘導型エンドヌクレアーゼをコードする核酸を含むベクター、およびRNA誘導型エンドヌクレアーゼ+ガイドRNAを含むタンパク質−RNA複合体を提供する。
本開示の別の面は、CRISPR/Cas様タンパク質またはそのフラグメントおよびエフェクタードメインを含む融合タンパク質を提供する。CRISPR/Cas様タンパク質は、ガイドRNAによって標的部位へ誘導され、その部位で、エフェクタードメインは、標的化核酸配列を修飾するか、またはそれに作用し得る。エフェクタードメインは、切断ドメイン、後成的修飾ドメイン、転写活性化ドメイン、または転写リプレッサードメインであり得る。融合タンパク質は、核局在化シグナル、細胞透過性ドメイン、またはマーカードメインから選択される少なくとも1つのさらなるドメインをさらに含み得る。
融合タンパク質は、CRISPR/Cas様タンパク質またはそのフラグメントを含む。CRISPR/Cas様タンパク質は、上記のセクション(I)に詳述している。CRISPR/Cas様タンパク質は、N末端、C末端、または融合タンパク質の内部に位置していてよい。
融合タンパク質は、エフェクタードメインもまた含む。エフェクタードメインは、切断ドメイン、後成的修飾ドメイン、転写活性化ドメイン、または転写リプレッサードメインであり得る。エフェクタードメインは、N末端、C末端、または融合タンパク質の内部に位置していてよい。
ある態様において、エフェクタードメインは切断ドメインである。本明細書で用いられている“切断ドメイン”は、DNAを切断するドメインを意味する。切断ドメインは、エンドヌクレアーゼまたはエキソヌクレアーゼから得られ得る。切断ドメインが由来し得るエンドヌクレアーゼの非限定的例には、制限エンドヌクレアーゼおよびホーミングエンドヌクレアーゼが含まれるが、これに限定されない。例えば、New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res. 25:3379−3388を参照のこと。DNAを切断するさらなる酵素が公知である(例えば、S1ヌクレアーゼ;マングビーンヌクレアーゼ;膵臓由来のDNase I;ミクロコッカルヌクレアーゼ;酵母のHO エンドヌクレアーゼ)。また、Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993を参照のこと。これらの酵素(またはその機能的フラグメント)の1つまたはそれ以上は、切断ドメインの供給源として使用可能である。
他の態様において、融合タンパク質のエフェクタードメインは、後成的修飾ドメインであってよい。一般的に、後成的修飾ドメインは、DNA配列を改変することなく、ヒストン構造および/または染色体構造を変化させる。変化したヒストンおよび/またはクロマチン構造は、遺伝子発現の変化をもたらすことができる。後成的修飾の例には、ヒストンタンパク質中のリシン残基のアセチル化またはメチル化、およびDNA中のシトシン残基のメチル化が含まれるが、これらに限定されない。好適な後成的修飾ドメインの非限定的な例には、ヒストンアセチルトランスフェラーゼドメイン、ヒストン脱アセチル化酵素ドメイン、ヒストンメチルトランスフェラーゼドメイン、ヒストン脱メチル化酵素ドメイン、DNAメチルトランスフェラーゼドメイン、およびDNA脱メチル化酵素ドメインが含まれる。
他の態様において、融合タンパク質のエフェクタードメインは転写活性化ドメインであってよい。一般的に、転写活性化ドメインは、転写調節因子および/または転写調節タンパク質(すなわち、転写因子、RNAポリメラーゼなど)と相互作用して、遺伝子の転写を増加および/または活性化する。ある態様において、転写活性化ドメインは、ヘルペス単純ウイルス VP16 活性化ドメイン、VP64(VP16の四量体誘導体である)、NFκB p65 活性化ドメイン、p53 活性化ドメイン1および2、CREB(cAMP応答因子結合タンパク質)活性化ドメイン、E2A活性化ドメイン、およびNFAT(活性化T細胞の核因子)活性化ドメインであり得るが、これに限定されない。他の態様において、転写活性化ドメインは、Gal4、Gcn4、MLL、Rtg3、Gln3、Oaf1、Pip2、Pdr1、Pdr3、Pho4、およびLeu3であり得る。転写活性化ドメインは野生型であってよく、または、オリジナルの転写活性化ドメインの改変体であってもよい。ある態様において、融合タンパク質のエフェクタードメインは、VP16またはVP64転写活性化ドメインである。
さらに他の態様において、融合タンパク質のエフェクタードメインは、転写リプレッサードメインであってよい。一般的に、転写リプレッサードメインは、転写調節因子および/または転写調節タンパク質(すなわち、転写因子、RNAポリメラーゼなど)に相互作用して、遺伝子の転写を減少および/または終了させる。好適な転写リプレッサードメインの非限定的な例には、cAMP誘導性初期リプレッサー(ICER)ドメイン、Kruppel付随ボックスA(KRAB−A)リプレッサードメイン、YY1 グリシン富化リプレッサードメイン、Sp1様リプレッサー、E(spl)リプレッサー、IκBリプレッサー、およびMeCP2が含まれる。
ある態様において、融合タンパク質は、少なくとも1つのさらなるドメインをさらに含む。好適なさらなるドメインの非限定的な例には、核局在化シグナル、細胞透過性ドメインまたは転移ドメイン、およびマーカードメインが含まれる。好適な核局在化シグナル、細胞透過性ドメイン、およびマーカードメインの非限定的な例は、上記のセクション(I)に記載されている。
融合タンパク質のエフェクタードメインが切断ドメインである態様において、少なくとも1つの融合タンパク質を含む二量体を形成可能である。該二量体は、ホモ二量体またはヘテロ二量体であり得る。ある態様において、ヘテロ二量体は、2つの異なる融合タンパク質を含む。他の態様において、ヘテロ二量体は、1つの融合タンパク質とさらなるタンパク質を含む。
本開示の別の面は、上記セクション(I)および(II)にそれぞれ詳述したRNA誘導型エンドヌクレアーゼまたは融合タンパク質の何れかをコードする核酸を提供する。該核酸は、RNAまたはDNAであり得る。一態様において、RNA誘導型エンドヌクレアーゼまたは融合タンパク質をコードする核酸は、mRNAである。mRNAは、5’キャップ付加および/または3’ポリアデニル化されていてよい。別の態様において、RNA誘導型エンドヌクレアーゼまたは融合タンパク質をコードする核酸はDNAである。DNAはベクター中に存在していてよい(下記参照)。
本開示の別の面は、真核細胞または胚において染色体配列を修飾するための方法を包含する。該方法は、真核細胞または胚に、(i)少なくとも1つの核局在化シグナルを含む少なくとも1つのRNA誘導型エンドヌクレアーゼまたは少なくとも1つの核局在化シグナルを含む少なくとも1つのRNA誘導型エンドヌクレアーゼをコードする核酸、(ii)少なくとも1つのガイドRNAまたは少なくとも1つのガイドRNAをコードするDNA、および、要すれば(iii)ドナー配列を含む少なくとも1つのドナーポリヌクレオチドを導入することを含む。該方法は、各ガイドRNAが、染色体配列中の標的部位にRNA誘導型エンドヌクレアーゼを誘導し、そこでRNA誘導型エンドヌクレアーゼが、標的部位における二本鎖の切断を誘導し、二本鎖の切断が、染色体配列が修飾されるようにDNA修復過程により修復されるように、細胞または胚を培養することをさらに含む。
本方法は、細胞または胚に、少なくとも1つの核局在化シグナルを含む少なくとも1つのRNA誘導型エンドヌクレアーゼまたは少なくとも1つの核局在化シグナルを含む少なくとも1つのRNA誘導型エンドヌクレアーゼをコードする核酸を導入することを含む。かかるRNA誘導型エンドヌクレアーゼおよびRNA誘導型エンドヌクレアーゼをコードする核酸は、上記のセクション(I)および(III)にそれぞれ記載されている。
本方法はまた、細胞または胚に、少なくとも1つのガイドRNAまたは少なくとも1つのガイドRNAをコードするDNAを導入することも含む。ガイドRNAは、RNA誘導型エンドヌクレアーゼと相互作用して、エンドヌクレアーゼを、染色体配列中の特定のプロトスペーサー配列を有するガイドRNA塩基対の5’末端である特定の標的部位へ導く。
ガイドRNAと結合させたRNA誘導型エンドヌクレアーゼは、染色体配列中の標的部位に導かれ、そこで、RNA誘導型エンドヌクレアーゼは、染色体配列中に二本鎖の切断を導入する。標的部位は、配列がコンセンサス配列の直前(上流)に位置することを除いて、配列に何らの制限もない。このコンセンサス配列は、プロトスペーサー近接モチーフ(PAM)としても知られている。PAMの例には、NGG、NGGNG、およびNNAGAAW(ここで、Nは何れかのヌクレオチドを意味し、WはAまたはTを意味する)が含まれるが、これに限定されない。上記のセクション(IV)(b)に詳述の通り、ガイドRNAの第一の領域(5’末端)は、標的配列のプロトスペーサーに相補的である。典型的に、ガイドRNAの第一の領域は、約19から21ヌクレオチド長である。従って、ある面において、染色体配列中の標的部位の配列は、5’−N19−21−NGG−3’である。PAMは、イタリック体(NGG)である。
ある態様において、本方法は、胚内に少なくとも1つのドナーポリヌクレオチドを導入することをさらに含む。ドナーポリヌクレオチドは、少なくとも1つのドナー配列を含む。ある面において、ドナーポリヌクレオチドのドナー配列は、内在性または天然の染色体配列に相当する。例えば、ドナー配列は、標的部位で、またはその近位で染色体配列の一部と実質的に同一であり得るが、少なくとも1つのヌクレオチド変化を含む。従って、ドナー配列は、天然の配列への組み込みまたはその置換によって、標的染色***置での配列が少なくとも1つのヌクレオチド変更を含むように、標的部位で野生型配列の変形を含み得る。例えば、該変更は、1またはそれ以上のヌクレオチドの挿入、1またはそれ以上のヌクレオチドの欠失、1またはそれ以上のヌクレオチドの置換、またはそれらの組み合わせであり得る。修飾された配列の挿入の結果として、細胞または胚/動物は、標的の染色体配列から改変された遺伝子産物を生成し得る。
ある態様において、ドナーポリヌクレオチド中のドナー配列は、染色体配列中の標的部位の、それぞれ上流および下流に位置する配列と実質的に同一の配列を有する、上流配列および下流配列により挟まれている。これらの配列が類似であるため、ドナーポリヌクレオチドの上流および下流配列は、ドナー配列が染色体配列中に組み込まれ得る(または置換可能な)ように、ドナーポリヌクレオチドと標的染色体配列の相同組換えを可能にする。
他の態様において、ドナーポリヌクレオチドは、RNA誘導型エンドヌクレアーゼにより認識される少なくとも1つの標的切断部位をさらに含み得る。ドナーポリヌクレオチドに付加された標的切断部位は、ドナー配列の上流もしくは下流またはその上流および下流の両方に位置し得る。例えば、ドナー配列は、標的切断部位に隣接することが可能であり、すなわち、RNA誘導型エンドヌクレアーゼによる切断によって、該ドナー配列が、RNA誘導型エンドヌクレアーゼによる切断によって生じる染色体配列中のドナー配列と適合性のオーバーハングにより連結されている。従って、ドナー配列は、非相同的修復過程による二本鎖切断の修復中に切断された染色体配列と結合され得る。一般的に、標的切断部位(複数可)を含むドナーポリヌクレオチドは、環状であり得る(例えば、プラスミドベクターの一部であってよい)。
さらに別の態様において、ドナーポリヌクレオチドは、RNA誘導型エンドヌクレアーゼによって生じるオーバーハングと適合する任意の短いオーバーハングを有する短いドナー配列を含む直鎖状分子であり得る。かかる態様において、ドナー配列は、二本鎖の切断の修復中に切断された染色体配列と直接結合され得る。ある例において、ドナー配列は、約1,000未満、約500未満、約250未満、または約100ヌクレオチド未満であり得る。ある場合には、ドナーポリヌクレオチドは、平滑末端を有する短いドナー配列を含む直鎖状分子であり得る。他の例(iteration)において、ドナーポリヌクレオチドは、5’および/または3’オーバーハングを有する短いドナー配列を含む直鎖状分子であり得る。オーバーハングは、1、2、3、4または5ヌクレオチドを含んでいてよい。
RNA標的化エンドヌクレアーゼ(複数可)(またはそれをコード化する核酸)、ガイドRNA(複数可)(またはそれをコード化するDNA)、および任意のドナーポリヌクレオチド(複数可)は、種々の手段により細胞または胚に導入され得る。ある態様において、該細胞または胚は、トランスフェクトされる。好適なトランスフェクション法には、リン酸カルシウム仲介性トランスフェクション、ヌクレオフェクション(または、エレクトロポレーション)、陽イオン性ポリマーを用いるトランスフェクション(例えば、DEAE−デキストランまたはポリエチレンイミン)、ウイルス形質導入、ビロソーム(virosome)を用いるトランスフェクション、ビリオンを介するトランスフェクション、リポソームトランスフェクション、陽イオン性リポソームを用いるトランスフェクション、免疫リポソーム(immunoliposome)を用いるトランスフェクション、非リポソーム系脂質を用いるトランスフェクション、デンドリマートランスフェクション、熱ショックトランスフェクション、マグネトフェクション、リポフェクション、遺伝子銃送達、インペールフェクション、ソノポレーション、光学トランスフェクション、および特殊薬物存在下の核酸取り込みが含まれる。トランスフェクション法は、当技術分野で公知である(例えば、“Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 または “Molecular Cloning: A Laboratory Manual” Sambrook & Russell、Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001を参照のこと)。他の態様において、分子は、マイクロインジェクションにより細胞または胚に導入される。典型的に、胚は、目的の種の受精後の1細胞期胚である。例えば、分子は、注入によって1細胞胚の前核に注入することができる。
本方法は、ガイドRNA(複数可)がRNA誘導型エンドヌクレアーゼ(複数可)を染色体配列中の標的部位(複数可)に導き、RNA誘導型エンドヌクレアーゼ(複数可)が染色体配列中に少なくとも1つの二本鎖の切断を導入するのに適当な条件下で、細胞または胚を維持することをさらに含む。二本鎖切断は、染色体配列が、少なくとも1つのヌクレオチドの欠失、少なくとも1つのヌクレオチドの挿入、少なくとも1つのヌクレオチドの置換、またはそれらの組み合わせにより修飾されるように、DNA修復過程により修復され得る。
広範囲の真核細胞および胚が、本方法における使用に好適である。例えば、細胞は、ヒト細胞、非ヒト哺乳動物細胞、非哺乳動物脊椎動物細胞、無脊椎動物細胞、昆虫細胞、植物細胞、酵母細胞、または単細胞真核生物であり得る。一般的に、胚は、非ヒト哺乳動物胚である。特定の態様において、胚は、1つの細胞の非ヒト哺乳動物胚であり得る。1つの細胞胚を含む哺乳動物胚の例には、マウス胚、ラット胚、ハムスター胚、げっ歯動物胚、ウサギ胚、ネコ胚、ヒツジ胚、ブタ胚、ウシ胚、ウマ胚、および霊長動物胚が含まれるが、これに限定されない。さらに他の態様において、細胞は幹細胞であり得る。好適な幹細胞には、胚性幹細胞、ES様幹細胞、胎生幹細胞、成体幹細胞、多能性幹細胞、誘導多能性幹細胞、多能性(multipotent)幹細胞、少能性(oligopotent)幹細胞、および単能性(unipotent)幹細胞等が含まれるが、これらに限定されない。例示的態様において、細胞は哺乳動物細胞である。
本開示の別の面は、細胞または胚において、染色体配列を修飾するか、または染色体配列の発現を制御するための方法を包含する。本方法は、細胞または胚へ、(a)少なくとも1つの融合タンパク質または少なくとも1つの融合タンパク質をコード化する核酸(ここで、該融合タンパク質は、CRISPR/Cas様タンパク質またはそのフラグメントおよびエフェクタードメインを含む)、および(b)少なくとも1つのガイドRNAまたはガイドRNAをコード化するDNA(ここで、該ガイドRNAは、融合タンパク質のCRISPR/Cas様タンパク質を染色体配列中の標的部位に誘導し、融合タンパク質のエフェクタードメインが染色体配列を修飾するか、または染色体配列の発現を制御する。)を導入することを含む。
本開示は、RNA誘導型エンドヌクレアーゼにより仲介されるか、または融合タンパク質により仲介される過程を用いて、例えば、本明細書に記載の方法を用いて修飾されている、少なくとも1つの染色体配列を含む遺伝子的に修飾された細胞、非ヒト胚および非ヒト動物を包含する。本開示は、目的の染色体配列または融合タンパク質、少なくとも1つのガイドRNA、および所望により、1つまたはそれ以上のドナーポリヌクレオチド(複数可)に対して標的化されたRNA誘導型エンドヌクレアーゼまたは融合タンパク質をコード化する少なくとも1つのDNAまたはRNA分子を含む細胞を提供する。本開示はまた、目的の染色体配列、少なくとも1つのガイドRNA、および所望により、1つまたはそれ以上のドナーポリヌクレオチド(複数可)に対して標的化されたRNA誘導型エンドヌクレアーゼまたは融合タンパク質をコード化する少なくとも1つのDNAまたはRNA分子を含む非ヒト胚を提供する。
特に他に定義されない限り、本明細書で用いる全ての技術用語および科学用語は、本発明の属する分野の当業者に通常理解される意味を有する。以下の参考文献は、本発明に使用される多くの用語の一般的定義を当業者に提供する:Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); および、 Hale & Marham, The Harper Collins Dictionary of Biology (1991)。本明細書で用いる通り、以下の用語は、他に特に記載されない限り、それらの有する普通の意味を有する。
以下の実施例は、本発明のある面を説明する。
化膿性連鎖球菌株MGAS15252(受託番号YP_005388840.1)由来のCas9遺伝子を、哺乳動物細胞におけるその翻訳を強化するためにヒト(ホモ・サピエンス)のコドンで優先的に最適化した。Cas9遺伝子はまた、哺乳動物細胞の核に該タンパク質を標的化するためにC末端に核局在化シグナルPKKKRKV(配列番号1)を付加することにより修飾された。表1は、修飾されたCas9アミノ酸配列を示し、核局在化配列は下線で示される。表2は、コドンを最適化した修飾されたCas9 DNA配列を示す。
アデノ随伴ウイルス挿入部位1(AAVS1)遺伝子座を、Cas9仲介性ヒトゲノム修飾のための標的として用いた。ヒト AAVS1遺伝子座は、タンパク質ホスファターゼ1、調節サブユニット12C(PPP1R12C)のイントロン1(4427bp)に位置する。表3は、PPP1R12Cの第一エクソン(網掛け灰色部分)および第一イントロンを示す。イントロン内の下線を付した配列は、標的修飾部位(すなわち、AAVS1遺伝子座)である。
PPP1R12CのN末端へのGFPタンパク質の特異的挿入を、Cas9仲介性ゲノム修飾をモニターするために用いた。相同組換えによる挿入を仲介するために、ドナーポリヌクレオチドを調製した。AAVS1−GFP DNA ドナーは、5’(1185bp)のAAVS1遺伝子座の相同アーム、RNAスプライシング受容体、ターボGFPコーディング配列、3’転写ターミネーター、および3’(1217bp)のAAVS1遺伝子座の相同アームを含んでいた。表5は、RNAスプライシング受容体の配列および3’転写ターミネーターが続くGFPコーディング配列を示す。プラスミドDNAを、GenEluteエンドトキシンフリープラスミドMaxiprep Kit(Sigma)を用いて調製した。
トランスフェクションをヒトK562細胞で行った。K562細胞株をAmerican Type Culture Collection (ATCC)より入手し、10%FBSおよび2mMのL−グルタミンを添加したイスコフ改変ダルベッコ培地中で増殖させた。全ての培地および添加物は、Sigma−Aldrichより入手した。培養物をトランスフェクションの1日前に(1mL当たり約50万個の細胞で)分割した。細胞を、T−016プログラムを用いてNucleofector (Lonza)のNucleofector ソリューションV(Lonza)を用いてトランスフェクションした。各ヌクレオフェクション(nucleofection)溶液は、約60万個の細胞を含んだ。トランスフェクション処理を表7に詳述する。細胞を、ヌクレオフェクション直後に37℃および5% CO2で増殖させた。
ゲノムDNAを、トランスフェクションの12日後にGenElute Mammalian Genomic DNA Miniprep Kit (Sigma)を用いてトランスフェクション細胞から抽出した。その後、ゲノムDNAを、AAVS1−GFPプラスミドドナーの5’相同アームの外側に位置するフォワードプライマーおよびGFPの5’領域に位置するリバースプライマーを用いて、PCR増幅させた。フォワードプライマーは、5’−CCACTCTGTGCTGACCACTCT−3’(配列番号18)であり、リバースプライマーは、5’−GCGGCACTCGATCTCCA−3’(配列番号19)であった。ジャンクションPCRから予期されるフラグメントサイズは、1388bpであった。増幅を、以下のサイクル条件を用いてJumpStart Taq ReadyMix (Sigma)を用いて行った:最初の変性のために98℃で2分間;98℃で15秒間、62℃で30秒間、および72℃で1分30秒を35サイクル;そして、最後の伸張を72℃で5分間。PCR産物を1%アガロースゲル上で分離した。
マウスRosa26遺伝子座を、ゲノム編集のための標的とし得る。表8は、マウスRosa26配列の一部を示し、そこで、可能性のある標的部位を太字で示す。各標的部位は、プロトスペーサーを含む。
Rosa26遺伝子座は、上記のセクション(IV)(d)に詳述した通り、ドナーポリヌクレオチドを上記の実施例6に記載のプレアニーリングしたcrRNA/tracrRNAおよび修飾されたCas9タンパク質をコード化するmRNAと共にインジェクションすることによりマウス胚において修飾され得る。生産動物由来のインビトロでインキュベートされた胚または組織(実施例6に記載)を、PCRに基づく遺伝子型決定法、例えばRFLPアッセイ、ジャンクションPCR、およびDNA配列決定等を用いて修飾されたRosa26遺伝子座についてスクリーニングすることができる。
ラットRosa26遺伝子座を、ゲノム修飾の標的とすることができる。表10は、ラット配列の一部を示し、そこで、可能性のある標的部位は太字で示される。各標的部位はプロトスペーサーを含む。
Rosa26遺伝子座は、上記のセクション(IV)(d)に詳述した通り、ドナーポリヌクレオチドを上記の実施例8に記載のプレアニーリングしたcrRNA/tracrRNAおよび修飾されたCas9をコード化するmRNAと共にインジェクションすることによりラット胚において修飾され得る。生産ラット由来のインビトロでインキュベートされた胚または組織(実施例8に記載)を、PCRに基づく遺伝子型決定法、例えばRFLPアッセイ、ジャンクションPCR、およびDNA配列決定等を用いて修飾されたRosa26遺伝子座についてスクリーニングすることができる。
Claims (21)
- 2つのRNA誘導型ニッカーゼシステムを含む組成物であって、各々のRNA誘導型ニッカーゼシステムが、(i)1つの機能的ヌクレアーゼドメインを有し、二本鎖配列の一本鎖を切断するように修飾されたRNA誘導型エンドヌクレアーゼ、ここで、RNA誘導型エンドヌクレアーゼが、II型のクラスタ化調節的散在型短パリンドローム反復配列(CRISPR)/CRISPR−関連(Cas)(CRISPR/Cas)システムタンパク質に由来し、II型CRISPR/CasシステムがCas9タンパク質である、および(ii)二本鎖配列の一本鎖中の標的部位と相補性を有する第一の領域およびRNA誘導型エンドヌクレアーゼと相互作用する第二の領域を含むガイドRNA、を含み、2つのRNA誘導型ニッカーゼシステムの標的部位が二本鎖配列の対向する鎖にあり、各々の標的部位がプロトスペーサー近接モチーフ(PAM)の直前に位置し、2つのRNA誘導型ニッカーゼシステムのRNA誘導型エンドヌクレアーゼが共に、二本鎖配列の対向する鎖を独立に切断することにより、二本鎖の切断を導入できるものである、組成物。
- 各々のRNA誘導型ニッカーゼシステムのCas9タンパク質が、RuvCまたはHNHドメイン中の変異を含む、請求項1に記載の組成物。
- 各々のRNA誘導型エンドヌクレアーゼが、細胞透過性ドメインまたはマーカードメインから選択される少なくとも1つの追加のドメインをさらに含む、請求項1または2に記載の組成物。
- ガイドRNAが少なくとも部分的に化学的に合成された、請求項1から3のいずれか一項に記載の組成物。
- 請求項1から4のいずれか一項に記載の組成物をコードする複数の核酸。
- 各々のRNA誘導型エンドヌクレアーゼをコードする核酸がmRNAであり、各々のガイドRNAをコードする核酸がDNAである、請求項5に記載の複数の核酸。
- 各々のRNA誘導型エンドヌクレアーゼをコードする核酸がDNAであり、各々のガイドRNAをコードする核酸がDNAである、請求項5に記載の複数の核酸。
- 各々のRNA誘導型エンドヌクレアーゼをコードする核酸が、真核細胞における発現のためにコドン最適化されている、請求項6または7に記載の複数の核酸。
- 請求項5から8のいずれか一項に記載の複数の核酸を含む少なくとも一つのベクターであって、各々のRNA誘導型エンドヌクレアーゼをコードする核酸が、真核細胞における発現のためにプロモーター調節配列に操作可能に連結されており、各々のガイドRNAをコードする核酸が、真核細胞における発現のためにプロモーター調節配列に操作可能に連結されている、ベクター。
- 各々のRNA誘導型エンドヌクレアーゼをコードする核酸が、真核細胞における発現のためにコドン最適化されている、請求項9に記載のベクター。
- 真核細胞において二本鎖配列を修飾するための方法(人間を手術、治療または診断する方法を除く)であって、該方法は、
a)真核細胞に、2つのRNA誘導型ニッカーゼシステムまたは該システムをコードする核酸、および任意にドナーポリヌクレオチド、ここで、各々のRNA誘導型ニッカーゼシステムが:
(i)1つの機能的ヌクレアーゼドメインを有し、二本鎖配列の一本鎖を切断するように修飾されたRNA誘導型エンドヌクレアーゼ、ここで、RNA誘導型エンドヌクレアーゼが、II型のクラスタ化調節的散在型短パリンドローム反復配列(CRISPR)/CRISPR−関連(Cas)(CRISPR/Cas)システムタンパク質に由来し、II型CRISPR/CasシステムがCas9タンパク質である;および
(ii)二本鎖配列の一本鎖中の標的部位と相補性を有する第一の領域およびRNA誘導型エンドヌクレアーゼと相互作用する第二の領域を含むガイドRNA、ここで、2つのRNA誘導型ニッカーゼシステムの標的部位が二本鎖配列の対向する鎖にあり、各々の標的部位がプロトスペーサー近接モチーフ(PAM)の直前に位置する;
を導入すること、および
b)2つのRNA誘導型ニッカーゼシステムのRNA誘導型エンドヌクレアーゼが共に、二本鎖配列の対向する鎖を独立に切断することにより、二本鎖の切断を導入し、DNA修復過程による二本鎖の切断の修復が、二本鎖配列の修飾を導入するように、真核細胞を培養すること、
を含む方法。 - 各々のRNA誘導型ニッカーゼシステムのCas9タンパク質が、RuvCまたはHNHドメイン中の変異を含む、請求項11に記載の方法。
- 各々のRNA誘導型エンドヌクレアーゼが、核局在化シグナル、細胞透過性ドメイン、またはマーカードメインから選択される少なくとも1つの追加のドメインをさらに含む、請求項11または12に記載の方法。
- 少なくとも1つのガイドRNAが少なくとも部分的に化学的に合成された、請求項11から13のいずれか一項に記載の方法。
- 任意のドナーポリヌクレオチドが、二本鎖配列における標的部位の近位の二本鎖配列と関連する少なくとも一つのヌクレオチド変化を含むドナー配列を含む、請求項11から14のいずれか一項に記載の方法。
- ドナー配列が、二本鎖染色体配列中の標的部位のどちらかの側の配列と実質的に同一の配列を有する配列と隣接しているか、またはRNA誘導型エンドヌクレアーゼによって生じるオーバーハングと適合する短いオーバーハングと隣接している、請求項15に記載の方法。
- 各々のRNA誘導型エンドヌクレアーゼをコードする核酸がmRNAであり、各々のガイドRNAをコードする配列がDNAである、請求項11から16のいずれか一項に記載の方法。
- 各々のRNA誘導型エンドヌクレアーゼをコードする核酸がDNAであり、各々のガイドRNAをコードする配列がDNAである、請求項11から16のいずれか一項に記載の方法。
- 各々のRNA誘導型エンドヌクレアーゼおよび各々のガイドRNAをコードする核酸がベクターの一部であり、各々のRNA誘導型エンドヌクレアーゼをコードする核酸が、真核細胞における発現のためにプロモーター調節配列に操作可能に連結されており、各々のガイドRNAをコードする核酸が、真核細胞における発現のためにプロモーター調節配列に操作可能に連結されている、請求項11から18のいずれか一項に記載の方法。
- 各々のRNA誘導型エンドヌクレアーゼをコードする核酸が、真核細胞における発現のためにコドン最適化されている、請求項11から19のいずれか一項に記載の方法。
- 真核細胞が、ヒト細胞、非ヒト哺乳動物細胞、または植物細胞である、請求項11から20のいずれか一項に記載の方法。
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