JP6700788B2 - Rna誘導性ヒトゲノム改変 - Google Patents
Rna誘導性ヒトゲノム改変 Download PDFInfo
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- JP6700788B2 JP6700788B2 JP2015549528A JP2015549528A JP6700788B2 JP 6700788 B2 JP6700788 B2 JP 6700788B2 JP 2015549528 A JP2015549528 A JP 2015549528A JP 2015549528 A JP2015549528 A JP 2015549528A JP 6700788 B2 JP6700788 B2 JP 6700788B2
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Description
本願は、2013年3月13日に出願された米国特許仮出願第61/779,169号および2012年12月17日に出願された米国特許仮出願第61/738,355号に基づく優先権を主張するものであり、あらゆる目的のため、その全体が参照により本明細書に組み込まれる。
本発明は、国立衛生研究所により授与されたP50HG005550の下、政府の援助により成されたものである。政府は、本発明に関して一定の権利を有する。
(b)隣接するgRNA−Cas9複合体の二量体化に伴う「高度に特異的な」ゲノム編集を可能にするためのFokIなどのヌクレアーゼドメイン;
(c)ゲノム座位および 染色体動態を可視化するための蛍光タンパク質;または
(d)タンパク質または 核酸が結合した有機フルオロフォア、量子ドット、分子ビーコン(molecular beacon)、および エコープローブまたは 分子ビーコン代替物などのその他の蛍光分子;
(e)ゲノムワイドな3次元構造のプログラム可能な操作を可能にする多価リガンド結合タンパク質ドメイン。
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II型CRISPR−Casシステム
ある態様によれば、本開示の実施形態は、真核細胞におけるヌクレアーゼによる活性のため、外来核酸を同定するための短鎖RNAを用いる。本開示のある態様によれば、真核細胞は、1または 複数の短鎖RNAと標的DNA配列への短鎖RNAの結合により活性化される1または 複数のヌクレアーゼとをコードする核酸をそのゲノム内に含むように改変される。ある態様によれば、例示的な短鎖RNA/酵素システムは、例えば短鎖RNAを用いて外来核酸の分解を導く(CRISPR)/CRISPR関連(Cas)システムなど、細菌または古細菌中に同定され得る。CRISPR(「clustered regularly interspaced short palindromic repeats」)防御には、侵入ウイルスまたはプラスミドDNA由来の新規標的化「スペーサー」のCRISPR座位への獲得および 組み込み、スペーサー反復単位からなる短鎖ガイドCRISPR RNA(crRNA)の発現および プロセシング、ならびに スペーサーに相補的な核酸(最も一般的にはDNA)の切断が含まれる。
プラスミド構築
Cas9遺伝子配列をヒトコドン最適化し、IDT社に注文した9種の500bpのgBlockの階層的融合PCRアセンブリ(hierachical fusion PCR assembly)により構築した。ヒト細胞のための改変II型CRISPRシステムを示す図3Aは、cas9遺伝子挿入断片の発現フォーマットおよび完全配列を示す。RuvC様モチーフおよび HNHモチーフ、ならびに C末端SV40NLSはそれぞれ青色、茶色、および 橙色で強調されている。Cas9_D10Aを同様に構築した。得られた全長産物をpcDNA3.3−TOPOベクター(Invitrogen社)にクローニングした。標的gRNA発現コンストラクトは、個別の455bpのgBlockとしてIDT社に直接注文し、pCR−BluntII−TOPOベクター(Invitrogen社)にクローニングするかPCR増幅を行った。図3Bは、ガイドRNAのU6プロモーターに基づく発現スキームおよびRNA転写産物の予想二次構造を示している。U6プロモーターの使用により、RNA転写産物の1位が「G」に限定されるので、このアプローチを用いて、GN20GGの形態の全ゲノム部位を標的とすることができる。図3Cは、使用した7種のgRNAを示している。
細胞培養
PGP1 iPS細胞は、マトリゲル(BD Biosciences社)でコーティングされたプレート上、mTeSR1(Stemcell Technologies社)中で維持した。TrypLE Express(Invitrogen社)を用いて5〜7日毎に継代培養した。K562細胞は、15% FBS含有RPM I(Invitrogen社)中で培養および維持した。HEK293T細胞は、10% ウシ胎児血清(FBS、Invitrogen社)、ペニシリン/ストレプトマイシン(pen/strep、Invitrogen社)、および非必須アミノ酸(NEAA、Invitrogen社)を添加したダルベッコ変法イーグル培地(DMEM、Invitrogen社)高グルコース中で培養した。全ての細胞は、加湿したインキュベーター中、37℃、5% CO2で維持した。
PGP1 iPS、K562、および293Tの遺伝子標的化
PGP1 iPS細胞は、ヌクレオフェクションの2時間前にRhoキナーゼ(ROCK)阻害剤(Calbiochem社)中で培養した。TrypLE Express(Invitrogen社)を用いて細胞を回収し、2×106個の細胞を、1μgのCas9プラスミド、1μgのgRNA、および/または1μgのDNAドナープラスミドと共にP3試薬(Lonza社)に再懸濁し、製造業者の指示書(Lonza社)に従ってヌクレオフェクトした。続いて、細胞を、mTeSRlでコーティングされたプレート上で、ROCK阻害剤を添加したmTeSRl培地中に最初の24時間プレーティングした。K562では、2×106個の細胞を、1μgのCas9プラスミド、1μgのgRNA、および/または1μgのDNAドナープラスミドと共にSF試薬(Lonza社)に再懸濁し、製造業者の指示書(Lonza社)に従ってヌクレオフェクトした。293Tでは、製造業者のプロトコールに従い、Lipofectamine2000を用いて0.1×106個の細胞に1μgのCas9プラスミド、1μgのgRNA、および/または1μgのDNAドナープラスミドをトランスフェクトした。内在性AAVS1の標的化に用いたDNAドナーは、dsDNAドナー(図2C)または90塩基長のオリゴヌクレオチドであった。前者は、標的化に成功した細胞を濃縮するためのSA−2A−ピューロマイシン−CaGGS−eGFPカセット、および隣接する短い相同性アームを有する。
AAVS1−R:
CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTacaggaggtgggggttagac
AAVS1−F.1:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTGATtatattcccagggccggtta
AAVS1−F.2:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTACATCGtatattcccagggccggtta
AAVS1−F.3:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCTAAtatattcccagggccggtta
AAVS1−F.4:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGTCAtatattcccagggccggtta
AAVS1−F.5:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCACTGTtatattcccagggccggtta
AAVS1−F.6:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTATTGGCtatattcccagggccggtta
AAVS1−F.7:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGATCTGtatattcccagggccggtta
AAVS1−F.8:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAAGTtatattcccagggccggtta
AAVS1−F.9:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGATCtatattcccagggccggtta
AAVS1−F.10:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCTAtatattcccagggccggtta
AAVS1−F.11:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTAGCCtatattcccagggccggtta
AAVS 1−F.12:
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACAAGtatattcccagggccggtta
HR_AAVS1−F
CTGCCGTCTCTCTCCTGAGT
HR_Puro−R
GTGGGCTTGTACTCGGTCAT
ヒトエキソンのCRISPR標的を計算するためのバイオインフォマティクスアプローチおよびそれらの多重合成の方法論
ヒトエキソンにおいて特異的部位を最大限に標的とするが、ゲノム中の他の部位は最小限に標的とするgRNA遺伝子配列セットは、以下のように決定した。ある態様によれば、gRNAによる最大効率の標的化は23ヌクレオチド(nt)の配列により達成され、そのうちの最も5′側の20ヌクレオチドは所望の部位に正確に相補的であり、最も3′側の3塩基はNGGの形態でなければならない。さらに、pol−III転写開始部位を確立 するために、最も5′側のヌクレオチドはGでなければならない。しかし、参照文献(2)によれば、ゲノム標的に対する20bpのgRNAの最も5′側の6個のヌクレオチドの不対合は、最後の14ヌクレオチドが適切に対合する限りCas9を介した切断を抑制しないが、最も5′側の8個のヌクレオチドの不対合および 最後の12ヌクレオチドの対合は切断を抑制し、最も5′側の7個のヌクレオチドの不対合および 13個の3′側の対合の場合については試験されていない。オフターゲット効果に関して保存的(conservative)であるための条件の1つは、6個の場合同様に、最も5′側の7個の不対合の場合に切断が許容されることであり、したがって最も3′側の13ヌクレオチドの対合が切断には十分である。オフターゲット切断をせずに切断が可能であるヒトエキソン内のCRISPR標的部位を同定するために、5′−GBBBB BBBBB BBBBB BBBBB NGG−3′の形態(第1の形態)の23bpの配列の全てを調べた。式中、Bは、エキソン部位の塩基を表し、この配列に対して、ヒトゲノム中の他のどの部位にも5′−NNNNN NNBBB BBBBB BBBBB NGG−3′の形態(第2の形態)の配列は存在しない。具体的には、(i)UCSC Genome Browser(参照文献15〜17)からGRCh37/hg19ヒトゲノムの全てのRefSeq遺伝子のコード領域部位のBEDファイルをダウンロードした。このBEDファイル中のコードエキソンの部位は、hg19ゲノムへのRefSeq mRNAアクセッションの346089個のマッピングのセットで構成されていた。しかし、いくつかのRefSeq mRNAアクセッションは複数のゲノム部位にマッピングされており(おそらく遺伝子重複)、多くのアクセッションが同一セットのエキソン部位のサブセットにマッピングされていた(同じ遺伝子の複数のアイソフォーム)。明らかに重複した遺伝子インスタンスを区別して、複数のRefSeqアイソフォームアクセッションによる同一のゲノムエキソンインスタンスへの複数の参照を統合するために、(ii)複数のゲノム部位を有した705個のRefSeqアクセッション番号に固有の数値サフィックス(num erical suffix)を付与し、(iii)BEDTools(参照文献18)(v2.16.2−zip−87e3926)のmergeBed機能を用いて、重複するエキソン部位を、マージされたエキソン領域に統合した。これらのステップにより、最初の346089個のRefSeqエキソン部位セットは、192783個の別個のゲノム領域に減少した。UCSC Table Browserを用いて、全てのマージされたエキソン領域のhg19配列をダウンロードし、各末端に20bpのパディング(padding)を付加した。(iv)カスタムperlコードを用いて、このエキソン配列内に第1の形態の1657793個のインスタンスを同定した。(v)次いで、これらの配列を、第2の形態のオフターゲットの存在についてフィルタリングした。すなわち、各々のマージされたエキソンの第1の形態の標的につき、最も3′側の13bpの特異的(B)「コア」配列を抽出し、各コアについて、16bpの配列である5′−BBB BBBBB BBBBB NGG−3′(N=A、C、G、およびT)を4種作成し、−l 16 −v 0 −k 2のパラメーターを用いたBowtie version 0.12.8(参照文献19)を用いて、これら6631172個の配列への正確なマッチについてhg19ゲノム全体をサーチした。2個以上のマッチがあった任意のエキソン標的部位は不採用とした。任意の特異的な13bpのコア配列およびそれに続く配列NGGにより15bpの特異性が付与されるのみであるため、ランダムな約3Gbの配列(両鎖)中に、拡張コア配列に対して平均約5.6個のマッチがあるはずである。そのため、最初に同定された1657793個の標的のほとんどが不採用とされたが、189864個の配列がこのフィルターをパスした。これらは、ヒトゲノム中のCRISPRで標的化可能なエキソン部位のセットを含む。この189864個の配列は、標的エキソン領域当たり約2.4部位の多重度で、78028個のマージされたエキソン領域中の部位を標的とする(全192783個のマージされたヒトエキソン領域の約40.5% )。遺伝子レベルで標的化を評価するために、マージされたエキソン領域と重複する任意の2つのRefSeqアクセッション((ii)で区別した遺伝子重複物を含む)が単一遺伝子クラスターとしてカウントされるようにRefSeq mRNAマッピングをクラスター化すると、189864個のエキソン特異的CRISPR部位が、標的遺伝子クラスター当たり約11.1の多重度で、18872個の遺伝子クラスターのうちの17104個(全遺伝子クラスターの約90.6% )を標的とする。(これらの遺伝子クラスターは、単一の転写遺伝子の複数のアイソフォームを表すRefSeq mRNAアクセッションを単一のエンティティ(entity)へと折り畳んでいる(collapse)が、これらにはさらに、重複する別個の遺伝子およびアンチセンス転写産物を有する遺伝子も折り畳まれていることに留意されたい。)オリジナルのRefSeqアクセッションレベルでは、189864個の配列が、マッピングされたRefSeqアクセッション(区別される重複遺伝子を含む)の全43726個のうちの30563個(約69.9% )において、標的とするマッピングされたRefSeqアクセッション1個当たり約6.2部位の多重度で、エキソン領域を標的とした。
多重合成
標的配列を、DNAアレイ上での多重合成に適合する200bpフォーマットに組み込んだ(参照文献23、24)。ある態様によれば、この方法により、DNAアレイに基づくオリゴヌクレオチドプールからの特異的なgRNA配列またはgRNA配列プールの標的化された回収、および一般的な発現ベクターへの迅速なクローニングが可能になる(図13A)。具体的には、CustomArray Inc.社の12kのオリゴヌクレオチドプールを合成した。さらに、このライブラリーから、最適なgRNAの回収(図13B)に成功した。合成DNA1000bp当たりの誤り率は約4変異であった。
RNA誘導性ゲノム編集は標的化の成功のためにCas9および ガイドRNAの両方を必要とする
図1Bに記載のGFPレポーターアッセイを用いて、修復DNAドナー、Cas9タンパク質、および gRNAの全ての可能な組み合わせの、(293Tにおいて)HRを成功させる能力について試験した。図4に示すように、3つのコンポーネントの全てが存在する場合にのみGFP+細胞が観察され、これにより、RNA誘導性ゲノム編集にこれらのCRISPRコンポーネントが必須であることが確認された。データは平均値±SEM(N=3)である。
gRNAおよび Gas9を介したゲノム編集の分析
(A)図5Aに結果が示される前述のGFPレポーターアッセイおよび (B)図5Bに結果が示される(293Tにおける)標的部位のディープシークエンシングのいずれかを用いて、CRISPRを介したゲノム編集プロセスを調べた。比較として、以前の報告でインビトロアッセイにおいてニッカーゼとして機能することが示されているCas9のD10A変異体を試験した。図5に示すように、Cas9およびCas9D10Aはどちらもほぼ同じ率でHRを成功させることができた。しかし、ディープシークエンシングから、Cas9は標的部位で強固なNHEJを示すが、(DNAにニックを入れるだけの推定能力から予想される通り)D10A変異体はNHEJ比率が有意に低減していることが確認される。さらに、Cas9タンパク質の既知の生化学と一致して、NHEJデータから、塩基対の欠失または挿入の大部分が標的配列の3′末端で起こっており、ピークはPAM部位の上流約3〜4塩基であり、欠失頻度の中央値は約9〜10bpであることが確認される。データは平均値±SEM(N=3)である。
RNA誘導性ゲノム編集は標的配列特異的である
図1Bに記載のGFPレポーターアッセイ同様、別個のGFPレポーターコンストラクトをそれぞれ有する3種の293T安定発現細胞株を開発した。これらは(図6に示されるように)AAVS1断片インサートの配列によって区別される。1種の細胞株は野生型断片を有し、他の2種の細胞株は6bpが変異している(赤色で強調)。次いで、各細胞株を以下の4つの試薬、すなわち、標的配列が隣接GFP断片中にあるために全ての細胞株中に存在するので、全ての細胞型を標的化可能であるGFP−ZFNの組み合わせ;他の2種の細胞株における変異により左のTALENがそれらの部位に結合できなくなっていると考えられるので、wt−AAVS1断片のみを潜在的に標的化し得るAAVS1 TALEN;この標的部位も2種の変異細胞株中で中断されているので、やはりwt−AAVS1断片のみを潜在的に標的化可能であるT1gRNA;およびT1gRNAと異なり、標的部位が3種の細胞株で変化していないので、3種の細胞株の全てを標的化可能であると考えられるT2gRNAのいずれかで標的化した。ZFNは3種の細胞型全てを改変し、AAVS1 TALENお よびT1gRNAはwt−AAVS1細胞型のみを標的化し、T2gRNAは3種の細胞型全ての標的化に成功した。総合して、これらの結果により、ガイドRNAを介した編集が標的配列特異的であることが確認される。データは平均値±SEM(N=3)である。
GFP配列を標的とするガイドRNAは強固なゲノム編集を可能にする
AAVS1インサートを標的化する2種のgRNAに加えて、(293Tにおいて)図1Bに記載のレポーターの隣接GFP配列を標的化する2種の新たなgRNAを試験した。図7に示すように、これらのgRNAもまた、この改変された座位で強固なHRをもたらすことができた。データは平均値±SEM(N=3)である。
RNA誘導性ゲノム編集は標的配列特異的であり、ZFNまたはTALENと同様な標的化効率を示す
図1Bに記載のGFPレポーターアッセイ同様、別個のGFPレポーターコンストラクトをそれぞれ有する2種の293T安定発現細胞株を開発した。(図8に示すように)これらは断片インサートの配列によって区別される。一方の細胞株はDNMT3a遺伝子由来の58bp断片を有し、もう一方の細胞株はDNMT3b遺伝子由来の相同な58bp断片を有する。配列の差は赤色で強調される。次いで、各細胞株を以下の6つの試薬、すなわち、その標的配列が隣接GFP断片中にあるために全ての細胞株中に存在するので、全ての細胞型を標的化可能であるGFP−ZFNの組み合わせ;DNMT3a断片またはDNMT3b断片を潜在的に標的化するTALENの組み合わせ;DNMT3a断片のみを潜在的に標的化可能なgRNAの組み合わせ;およびDNMT3b断片のみを潜在的に標的化すると考えられるgRNAのいずれかで標的化した。図8に示すように、ZFNは3つの細胞型全てを改変し、TALENお よびgRNAはそれぞれの標的のみを改変した。さらに、標的化効率は6つの標的化試薬で同等であった。総合すると、これらの結果により、RNA誘導性編集が標的配列特異的であり、ZFNまたはTALENと同様な標的化効率を示すことが確認される。データは平均値±SEM(N=3)である。
ヒトiPS細胞におけるRNA誘導性NHEJ
ヒトiPS細胞(PGP1)に、図9の左パネルに示すコンストラクトをヌクレオフェクトした。ヌクレオフェクションの4日後、DNA二本鎖切断部(DSB)でのゲノムの欠失率および挿入率をディープシークエンシングにより評価することによってNHEJ比率を測定した。パネル1:標的領域で検出された欠失率。赤色の破線:T1RNA標的部位の境界;緑色の破線:T2RNA標的部位の境界。各ヌクレオチド位置での欠失の発生率を黒線にプロットし、欠失を有するリードの割合として欠失率を計算した。パネル2:標的領域で検出された挿入率。赤色の破線:T1RNA標的部位の境界;緑色の破線:T2RNA標的部位の境界。最初の挿入接合部が検出されたゲノム位置での挿入の発生率を黒線にプロットし、挿入を有するリードの割合として挿入率を計算した。パネル3:欠失サイズの分布。全NHEJ集団間の異なるサイズの欠失の頻度をプロットした。パネル4:挿入サイズの分布。全NHEJ集団間の異なるサイズの挿入の頻度をプロットした。両方のgRNAによるiPS標的化は効率的であり(2〜4% )、配列特異的であり(NHEJ欠失分布の位置のシフトにより示される)、図4の結果を再確認するものであり、NGSに基づく分析もまた、標的座位におけるNHEJ現象にCas9タンパク質およびgRNAの両方が必須であることを示している。
K562細胞におけるRNA誘導性NHEJ
図10の左パネルに示すコンストラクトをK562細胞に核化(nucleated)した。ヌクレオフェクションの4日後、DSBでのゲノムの欠失率および挿入率をディープシークエンシングにより評価することによってNHEJ比率を測定した。パネル1:標的領域で検出された欠失率。赤色の破線:T1RNA標的部位の境界;緑色の破線:T2RNA標的部位の境界。各ヌクレオチド位置での欠失の発生率を黒線にプロットし、欠失を有するリードの割合として欠失率を計算した。パネル2:標的領域で検出された挿入率。赤色の破線:T1RNA標的部位の境界;緑色の破線:T2RNA標的部位の境界。最初の挿入接合部が検出されたゲノム位置での挿入の発生率を黒線にプロットし、挿入を有するリードの割合として挿入率を計算した。パネル3:欠失サイズの分布。全NHEJ集団間の異なるサイズの欠失の頻度をプロットした。パネル4:挿入サイズの分布。全NHEJ集団間の異なるサイズの挿入の頻度をプロットした。両方のgRNAによるK562標的化は効率的であり(13〜38%)、配列特異的である(NHEJ欠失分布の位置のシフトにより示される)。重要なことに、観察された欠失サイズ頻度のヒストグラムにおけるピークからも明らかなように、T1ガイドRNAおよびT2ガイドRNAの同時導入により、その間に位置する19bp断片の高効率な欠失が生じており、このことは、このアプローチを用いてゲノム座位の多重編集も実現可能であることを示している。
293T細胞におけるRNA誘導性NHEJ
図11の左パネルに示すコンストラクトを293T細胞にトランスフェクトした。ヌクレオフェクションの4日後、DSBでのゲノムの欠失率および挿入率をディープシークエンシングにより評価することによってNHEJ比率を測定した。パネル1:標的領域で検出された欠失率。赤色の破線:T1RNA標的部位の境界;緑色の破線:T2RNA標的部位の境界。各ヌクレオチド位置での欠失の発生率を黒線にプロットし、欠失を有するリードの割合として欠失率を計算した。パネル2:標的領域で検出された挿入率。赤色の破線:T1RNA標的部位の境界;緑色の破線:T2RNA標的部位の境界。最初の挿入接合部が検出されたゲノム位置での挿入の発生率を黒線にプロットし、挿入を有するリードの割合として挿入率を計算した。パネル3:欠失サイズの分布。全NHEJ集団間の異なるサイズの欠失の頻度をプロットした。パネル4:挿入サイズの分布。全NHEJ集団間の異なるサイズの挿入の頻度をプロットした。両方のgRNAによる293T標的化は効率的であり(10〜24% )、配列特異的である(NHEJ欠失分布の位置のシフトにより示される)。
dsDNAドナーまたは短鎖オリゴヌクレオチドドナーのいずれかを用いた内在性AAVS1遺伝子座におけるHR
図12Aに示すように、PCRスクリーニング(図2Cを参照)により、21/24のランダムに選んだ293Tクローンで標的化の成功が確認された。図12Bに示すように、同様なPCRスクリーニングにより、3/7のランダムに選んだPGP1−iPSクローンでも標的化の成功が確認された。図12Cに示すように、90塩基長の短鎖オリゴもまた、内在性AAVS1遺伝子座での強固な標的化が可能であった(ここではK562細胞について示す)。
ヒトゲノム中の遺伝子を標的とするガイドRNAの多重合成、回収、およびU6発現ベクターのクローニングの方法
ヒトゲノム中の遺伝子の全エキソンの約40.5% を標的とする約190kのバイオインフォマティクス的に計算されたユニークなgRNA部位のリソースを作成した。図13Aに示すように、gRNA標的部位をDNAアレイ上での多重合成に適合する200bpフォーマットに組み込んだ。具体的には、このデザインにより、(i)DNAアレイオリゴヌクレオチドプールからの特異的gRNA標的またはgRNA標的のプールの(模式図に示すように、連続3ラウンドのネステッドPCRによる)標的化された回収;および(ii)AflIIを用いた直線化後にギブソン・ アセンブリを介したgRNAインサート断片の取込みのレシピエントとして機能する、一般的な発現ベクターへの迅速なクローニングが可能になる。図13Bに示すように、この方法を用いて、CustomArray社により合成された12kのオリゴヌクレオチドプールから10種のユニークなgRNAの標的化された回収が達成された。
CRISPRを介したRNA誘導性転写活性化
CRISPR−Casシステムは細菌の適応免疫防御システムを有し、侵入核酸を「切断する」ように機能する。ある態様によれば、CRISPR−CASシステムは、ヒト細胞において機能しゲノムDNAを「切断する」ように改変される。これは、(ヌクレアーゼ機能を有する)Cas9タンパク質をガイドRNA中のスペーサーに相補的な標的配列に導く短鎖ガイドRNAによって達成される。DNAを「切断する」能力により、ゲノム編集および標的化されたゲノム制御に関連する多くの応用が可能になる。このために、Cas9タンパク質を、(RuvC様ドメインおよびHNH様ドメインのヌクレアーゼ機能に重要であることが知られている)Mg2+へのカップリングを抑制すると予想される変異を導入することによりヌクレアーゼ欠損になるように変異させた。具体的には、D10A変異、D839A変異、H840A変異、およびN863A変異の組み合わせを導入した。(シークエンシング解析によりDNAを切断しない能力が確認された)このようにして作製されたCas9ヌクレアーゼ欠損タンパク質(以下、Cas9R−H−という)をその後、転写活性化ドメイン(ここではVP64)にカップリングして、CRISPR−casシステムがRNA誘導性転写制御因子として機能できるようにした(図14参照)。Cas9R−H−+VP64融合体は、示されている2つのレポーターにおいてRNA誘導性転写活性化を可能にする。具体的には、FACS分析および 免疫蛍光イメージングの両方により、このタンパク質が対応するレポーターのgRNA配列特異的標的化を可能にすること、および さらに、結果として起こる転写活性化が、dTomato蛍光タンパク質の発現によりアッセイされるように、従来のTALE−VP64融合タンパク質により誘導される転写活性化と同様のレベルであったことが示される。
gRNA配列の柔軟性および その応用
gRNAの5′部分、中央部分、および 3′部分における種々のランダムな配列挿入を体系的にアッセイすることにより、指定配列挿入(designer sequence insertion)に対するgRNA足場配列の柔軟性を決定した。具体的には、gRNA配列において、gRNAの5′末端、中央、および 3′末端に1bp、5bp、10bp、20bp、および 40bpのインサートを作製した(挿入の正確な位置は図15中で「赤色」で強調されている)。次いで、このgRNAの機能性を、(本明細書に記載されるように)GFPレポーターアッセイにおいてHRを誘導する能力により試験した。(保持されているHR誘導活性により測定されるように)5′末端および3′末端の配列挿入に対してgRNAが柔軟であることは明らかである。したがって、本開示の態様は、gRNA活性開始の引き金を引き得る小分子応答性RNAアプタマーのタグ化またはgRNAの可視化に関する。さらに、本開示の態様は、ハイブリダイゼーションによってssDNAドナーをgRNAに繋ぎ止めることに関し、これにより、ゲノム標的の切断と修復鋳型の即時の物理的局在化とのカップリングが可能になり、これにより誤りがちな非相同末端結合と比較して相同組換え率が促進される。
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Claims (9)
- ガイドRNAを真核細胞にトランスフェクトすること、および
前記ガイドRNAと相互作用するCas9タンパク質をコードする核酸を前記真核細胞にトランスフェクトすること
を含み、
前記ガイドRNAが前記真核細胞のゲノム上の標的核酸に結合し、
前記ガイドRNAが、スペーサーと前記スペーサーと連結された足場を含み、ここで、前記スペーサーは前記標的核酸に相補的であり、
前記足場が以下の核酸
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(配列番号:45)
を含み、
前記Cas9タンパク質が、ヌクレアーゼ欠損であり、前記ガイドRNAと相互作用し、前記標的核酸と結合し、
前記Cas9タンパク質にはFokIヌクレアーゼドメインが結合しており、
隣接するガイドRNA−Cas9タンパク質複合体のFokIヌクレアーゼドメインの二量体化により前記標的核酸が切断される、真核細胞をインビトロ又はエクスビボで改変する方法。 - 前記ガイドRNAが、前記ガイドRNAをコードする第一の核酸を前記真核細胞に導入することにより前記真核細胞にトランスフェクトされ、
前記Cas9タンパク質をコードする核酸が、前記Cas9タンパク質をコードする第二の核酸を前記真核細胞に導入することにより前記真核細胞にトランスフェクトされ、
前記真核細胞が、前記ガイドRNA及び前記Cas9タンパク質を生成する、請求項1に記載の方法。 - 前記第一の核酸が、ウイルス的送達法を用いて前記真核細胞に導入される、請求項2に記載の方法。
- 前記第一の核酸が、アデノ随伴ウイルスを用いて前記真核細胞に導入される、請求項2に記載の方法。
- 前記真核細胞が、酵母細胞、植物細胞、または哺乳動物細胞である、請求項1に記載の方法。
- 前記真核細胞がヒト細胞である、請求項1に記載の方法。
- 異なる標的核酸に相補的なスペーサーを有する複数のガイドRNAが、前記真核細胞にトランスフェクトされ、
前記複数のガイドRNAが、前記異なる標的核酸に結合し、
前記Cas9タンパク質が、前記複数のガイドRNAと相互作用する、請求項1に記載の方法。 - 前記Cas9タンパク質が、ヒトコドン最適化されている、請求項1に記載の方法。
- 前記Cas9タンパク質が、核局在化シグナルを含む、請求項1に記載の方法。
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