JP6896786B2 - 配列操作のためのCRISPR−Cas成分系、方法および組成物 - Google Patents
配列操作のためのCRISPR−Cas成分系、方法および組成物 Download PDFInfo
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Description
本出願は、それぞれBroad参照番号BI−2011/008/WSGR整理番号44063−701.101、BI−2011/008/WSGR整理番号44063−701.102、Broad参照番号BI−2011/008/VP整理番号44790.01.2003、BI−2011/008/VP整理番号44790.02.2003およびBI−2011/008/VP整理番号44790.03.2003を有する米国仮特許出願第61/736,527号明細書、同第61/748,427号明細書、同第61/768,959号明細書、同第61/791,409号明細書および同第61/835,931号明細書の優先権を主張し、これらは全て標題SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONであり、それぞれ2012年12月12日、2013年1月2日、2013年2月25日、2013年3月15日および2013年6月17日に出願されたものである。
米国仮特許出願第61/758,468号明細書;同第61/769,046号明細書;同第61/802,174号明細書;同第61/806,375号明細書;同第61/814,263号明細書;同第61/819,803号明細書および同第61/828,130号明細書が参照され、それぞれ、標題ENGINEERING AND OPTIMIZATION OF SYSTEMS,METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONであり、それぞれ、2013年1月30日;2013年2月25日;2013年3月15日;2013年3月28日;2013年4月20日;2013年5月6日および2013年5月28日に出願されたものである。それぞれ2013年6月17日に出願された米国仮特許出願第61/835,936号明細書、同第61/836,127号明細書、同第61/836,101号明細書、同第61/836,080号明細書、同第61/836,123号明細書および同第61/835,973号明細書も参照される。それぞれBroad参照番号BI−2011/008Aを有する米国仮特許出願第61/842,322号明細書および米国特許出願第14/054,414号明細書も参照され、標題CRISPR−CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTSを有し、それぞれ2013年7月2日および2013年10月15日に出願されたものである。
本発明は、米国国立衛生研究所(National Institutes of Health)により助成されたNIHパイオニアアワードDP1MH100706のもと政府支援によりなされた。米国政府は本発明において一定の権利を有する。
例示的なII型CRISPR系は、4つの遺伝子Cas9、Cas1、Cas2、およびCsn1のクラスター、ならびに2つの非コードRNAエレメント、tracrRNAおよび非反復配列の短いストレッチ(スペーサー、それぞれ約30bp)により間隔が空いている反復配列の特徴的アレイ(ダイレクトリピート)を含有する化膿性連鎖球菌(Streptococcus pyogenes)SF370からのII型CRISPR遺伝子座である。この系において、ターゲティングされるDNA二本鎖切断(DSB)を4つの連続ステップにおいて生成する(図2A)。第1に、2つの非コードRNA、プレcrRNAアレイおよびtracrRNAがCRISPR遺伝子座から転写される。第2に、tracrRNAがプレcrRNAのダイレクトリピートにハイブリダイズし、次いでそれが個々のスペーサー配列を含有する成熟crRNAにプロセシングされる。第3に、成熟crRNA:tracrRNA複合体がCas9を、crRNAのスペーサー領域とプロトスペーサーDNAとの間のヘテロ二本鎖形成を介してプロトスペーサーおよび対応するPAMからなるDNA標的に指向する。最後に、Cas9は、PAMの上流の標的DNAの開裂を媒介してプロトスペーサー内でDSBを創成する(図2A)。この例は、このRNAプログラマブルヌクレアーゼ系を適応させて真核細胞の核中のCRISPR複合体活性を指向する例示プロセスを記載する。
ヒト胚腎臓(HEK)細胞系HEK293FT(Life Technologies)を、10%のウシ胎仔血清(HyClone)、2mMのGlutaMAX(Life Technologies)、100U/mLのペニシリン、および100μg/mLのストレプトマイシンが補給されたダルベッコ改変イーグル培地(DMEM)中で37℃において5%のCO2インキュベーションで維持した。マウスneuro2A(N2A)細胞系(ATCC)を、5%のウシ胎仔血清(HyClone)、2mMのGlutaMAX(Life Technologies)、100U/mLのペニシリン、および100μg/mLのストレプトマイシンが補給されたDMEMにより、37℃、5%のCO2で維持した。
HEK293FTまたはN2A細胞を、上記プラスミドDNAにより形質移入した。形質移入後、細胞を37℃において72時間インキュベートしてからゲノムDNAを抽出した。ゲノムDNAは、QuickExtractDNA抽出キット(Epicentre)を製造業者のプロトコルに従って使用して抽出した。手短に述べると、細胞をQuickExtract溶液中で再懸濁させ、65℃において15分間および98℃において10分間インキュベートした。抽出されたゲノムDNAを直ちに処理または−20℃において貯蔵した。
HEK293FTおよびN2A細胞を、プラスミドDNAにより形質移入し、37℃において72時間インキュベートしてから上記のとおりゲノムDNAを抽出した。相同組換え(HR)テンプレートのホモロジーアーム外側のプライマーを使用して標的ゲノム領域をPCR増幅した。PCR産物を1%のアガロースゲル上で分離し、MinElute GelExtraction Kit(Qiagen)により抽出した。精製産物をHindIII(Fermentas)により消化し、6%のNovex TBEポリアクリルアミドゲル(Life Technologies)上で分析した。
RNA二次構造予測は、Institute for Theoretical Chemistry at the University of Viennaにおいて開発されたオンラインウェブサーバーRNAfoldを使用し、セントロイド構造予測アルゴリズムを使用して実施した(例えば、A.R.Gruber et al.,2008,Cell 106(1):23−24;およびPA Carr and GM Church,2009,Nature Biotechnology 27(12):1151−62参照)。
CRISPR活性に十分な化膿性連鎖球菌(S.pyogenes)CRISPR遺伝子座1のエレメントを、pCRISPRプラスミドを使用して大腸菌(E.coli)中で再構成した(図10Aに模式的に説明する)。pCRISPRは、tracrRNA、SpCas9、およびcrRNAアレイをドライブするリーダー配列を含有した。スペーサー(「ガイド配列」とも称される)を、説明のとおりアニールされたオリゴヌクレオチドを使用してcrRNAアレイ中にBsaI部位間で挿入した。干渉アッセイにおいて使用されるチャレンジプラスミドは、プロトスペーサー(「標的配列」とも称される)配列を、隣接CRISPRモチーフ配列(PAM)とともにpUC19中に挿入することにより構築した(図10B参照)。チャレンジプラスミドは、アンピシリン耐性を含有した。図10Cは、干渉アッセイの模式的表示を提供する。既にpCRISPRおよび適切なスペーサーを担持する化学コンピテント大腸菌(E.coli)株を、対応するプロトスペーサー−PAM配列を含有するチャレンジプラスミドにより形質転換した。pUC19を使用してそれぞれのpCRISPR担持コンピテント株の形質転換効率を評価した。CRISPR活性は、プロトスペーサーを担持するpPSPプラスミドの開裂をもたらし、そうでなければプロトスペーサーを欠くpUC19により付与されるアンピシリン耐性を除外した。図10Dは、図4Cに説明されるアッセイにおいて使用されたそれぞれのpCRISPR担持大腸菌(E.coli)株のコンピテンスを説明する。
HEK293FT細胞を上記のとおり維持および形質移入した。細胞をトリプシン処理により回収し、次いでリン酸緩衝生理食塩水(PBS)中で洗浄した。トータル細胞RNAをTRI試薬(Sigma)により製造業者のプロトコルに従って抽出した。抽出されたトータルRNAをNaonodrop(Thermo Scientific)を使用して定量し、同一濃度に正規化した。
RNAを等容量の2×ローディング緩衝液(Ambion)と混合し、95℃に5分間加熱し、氷上で1分間冷蔵し、次いで8%の変性ポリアクリルアミドゲル(SequaGel,National Diagnostics)上に、少なくとも30分間のゲルのプレラン後にロードした。試料を40W限界において1.5時間電気泳動した。その後、RNAをHybond N+メンブレン(GE Healthcare)に300mAにおいてセミドライ転写装置(Bio−rad)中で室温において1.5時間転写した。Stratagene UV CrosslinkerのStratalinker(Stratagene)上のオートクロスリンクボタンを使用してRNAをメンブレンに架橋させた。メンブレンをULTRAhyb−オリゴハイブリダイゼーション緩衝液(Ambion)中で回転させながら42℃において30分間プレハイブリダイズさせ、次いでプローブを添加し、一晩ハイブリダイズさせた。プローブはIDTに発注し、T4ポリヌクレオチドキナーゼ(New England Biolabs)を用いて[ガンマ−32P]ATP(Perkin Elmer)により標識した。メンブレンを予備加温(42℃)された2×SSC、0.5%のSDSにより1分間1回洗浄し、次いで42℃において30分間2回洗浄した。メンブレンを蛍光スクリーンに室温において1時間または一晩曝露させ、次いでphosphorimager(Typhoon)によりスキャンした。
tracrRNA、Cas9、およびリーダーを含むCRISPR遺伝子座エレメントを、化膿性連鎖球菌(Streptococcus pyogenes)SF370ゲノムDNAから、ギブソン・アセンブリ(Gibson Assembly)のためのフランキングホモロジーアームを用いてPCR増幅した。2つのBsaI IIS型部位を2つのダイレクトリピート間に導入してスペーサーの容易な挿入を促進した(図9)。Gibson Assembly Master Mix(NEB)を使用してPCR産物をEcoRV消化pACYC184中にtetプロモーターの下流でクローニングした。Csn2の最後の50bpは除き、他の内因性CRISPR系エレメントは除外した。相補的オーバーハングを有するスペーサーをコードするオリゴ(Integrated DNA Technology)をBsaI消化ベクターpDC000(NEB)中にクローニングし、次いでT7リガーゼ(Enzymatics)によりライゲートしてpCRISPRプラスミドを生成した。PAM配列(本明細書において「CRISPRモチーフ配列」とも称される)を有するスペーサーを含有するチャレンジプラスミドを、同等のオーバーハングを担持するハイブリダイズされたオリゴ(Integrated DNA Technology)をBamHI消化pUC19中にライゲートすることにより創成した。全ての構築物のためのクローニングは、大腸菌(E.coli)株JM109(Zymo Research)中で実施した。
配列特異的DNA開裂をプログラミングするためにRNAを使用する技能は、種々の研究および産業用途のための新たなクラスのゲノムエンジニアリングツールを定義する。CRISPR系のいくつかの態様は、CRISPRターゲティングの効率および多用途性を増加させるようにさらに改善することができる。最適なCas9活性は、哺乳動物核中に存在するものよりも高いレベルにおけるフリーMg2+の利用可能性に依存し得(例えば、Jinek et al.,2012,Science,337:816参照)、プロトスペーサーのすぐ下流のNGGモチーフについての優先性は、ヒトゲノム中で平均12bpごとでターゲティング能を制限する(図11、ヒト染色体配列のプラスおよびマイナス鎖の両方を評価)。これらの拘束の一部は、微生物メタゲノムにわたるCRISPR遺伝子座の多様性を利用することにより克服することができる(例えば、Makarova et al.,2011,Nat Rev Microbiol,9:467参照)。他のCRISPR遺伝子座を、実施例1に記載のものと同様の方法により哺乳動物細胞環境中に移植することができる。例えば、図12は、CRISPR媒介ゲノム編集を達成するための哺乳動物細胞中の異種発現のためのストレプトコッカス・サーモフィラス(Streptococcus thermophilus)LMD−9のCRISPR1からのII型CRISPR系の適応を説明する。図12Aは、S.サーモフィラス(S.thermophilus)LMD−9のCRISPR1の模式的説明を提供する。図12Bは、S.サーモフィラス(S.thermophilus)CRISPR系のための発現系の設計を説明する。ヒトコドン最適化hStCas9を、構成的EF1αプロモーターを使用して発現させる。tracrRNAおよびcrRNAの成熟バージョンを、U6プロモーターを使用して発現させて正確な転写開始を促進する。成熟crRNAおよびtracrRNAからの配列を説明する。crRNA配列中の小文字「a」により示される単一塩基を使用してRNApolIII転写ターミネーターとして機能するポリU配列を除去する。図12Cは、ヒトEMX1遺伝子座ターゲティングするガイド配列を示す模式図およびそれらの予測二次構造を提供する。それぞれの標的部位における改変効率を、RNA二次構造の下方に示す。この構造を生成するアルゴリズムは、それぞれの塩基を予測二次構造を仮定するその確率に従って着色し、これを図12Cにグレースケールで再現されるレインボースケールにより示す。図12Dは、Surveyorアッセイを使用する標的遺伝子座中のhStCas9媒介開裂の結果を示す。RNAガイドスペーサー1および2は、それぞれ14%および6.4%を誘導した。これらの2つのプロトスペーサー部位における生物学的複製物にわたる開裂活性の統計分析も図6に提供する。図16は、ヒトEMX1遺伝子座中のS.サーモフィラス(S.thermophilus)CRISPR系の追加のプロトスペーサーおよび対応するPAM配列標的の模式図を提供する。2つのプロトスペーサー配列を強調し、NNAGAAWモチーフを満たすそれらの対応するPAM配列を対応する強調配列に対して3’側で下線を付けることにより示す。両方のプロトスペーサーは、アンチセンス鎖をターゲティングする。
規定のCRISPR酵素についての所望のガイド配列長およびCRISPRモチーフ配列(PAM)に基づきインプットDNA配列の両方の鎖上の候補CRISPR標的配列を同定するためのソフトウェアプログラムを設計する。例えば、化膿性連鎖球菌(S.pyogenes)からのCas9についての標的部位は、PAM配列NGGを用いて、インプット配列およびインプットの逆相補鎖の両方の上の5’−Nx−NGG−3’を探索することにより同定することができる。同様に、S.サーモフィラス(S.thermophilus)CRISPR1のCas9についての標的部位は、PAM配列NNAGAAWを用いて、インプット配列およびインプットの逆相補鎖の両方の上の5’−Nx−NNAGAAW−3’を探索することにより同定することができる。同様に、S.サーモフィラス(S.thermophilus)CRISPR3のCas9についての標的部位は、PAM配列NGGNGを用いて、インプット配列およびインプットの逆相補鎖の両方の上の5’−Nx−NGGNG−3’を探索することにより同定することができる。Nx中の値「x」は、プログラムにより固定し、または使用者により規定することができ、例えば、20である。
本実施例は、異なる長さの野生型tracrRNA配列を取り込むtracr配列を有するキメラRNA(chiRNA;ガイド配列、tracrメイト配列、およびtracr配列を単一転写物中で含む)について得られた結果を記載する。図18aは、キメラRNAおよびCas9のためのバイシストロニック発現ベクターの模式図を説明する。Cas9はCBhプロモーターによりドライブされ、キメラRNAはU6プロモーターによりドライブされる。キメラガイドRNAは、からなる。示される種々の位置においてトランケートされたtracr配列(下方の鎖の最初の「U」から転写物の末端に及ぶ)に結合している20bpのガイド配列(N)からなる。ガイドおよびtracr配列は、tracrメイト配列GUUUUAGAGCUAと、それに続くループ配列GAAAにより離隔している。ヒト遺伝子座EMX1およびPVALB遺伝子座におけるCas9媒介インデルについてのSURVEYORアッセイの結果を、それぞれ図18bおよび18cに説明する。矢印は、予測SURVEYOR断片を示す。chiRNAをそれらの「+n」表記により示し、crRNAは、ガイドおよびtracr配列が別個の転写物として発現されるハイブリッドRNAを指す。トリプリケートで実施されたこれらの結果の定量を、図19aおよび19bにヒストグラムにより示し、それぞれ図18bおよび18cに対応する(「N.D.」は、インデルが検出されなかったことを示す)。プロトスペーサーIDおよびそれらの対応するゲノム標的、プロトスペーサー配列、PAM配列、および鎖局在を表Dに提供する。ガイド配列は、ハイブリッド系における別個の転写物の場合、プロトスペーサー配列全体に相補的であるように、またはキメラRNAの場合、下線部にのみ相補的であるように設計した。
ヒト胚腎臓(HEK)細胞系293FT(Life Technologies)を、10%のウシ胎仔血清(HyClone)、2mMのGlutaMAX(Life Technologies)、100U/mLのペニシリン、および100μg/mLのストレプトマイシンが補給されたダルベッコ改変イーグル培地(DMEM)中で37℃において5%のCO2インキュベーションで維持した。293FT細胞を24ウェルプレート(Corning)上に、形質移入24時間前に1ウェル当たり150,000個の細胞の密度において播種した。Lipofectamine2000(Life Technologies)を製造業者の推奨プロトコルに従って使用して細胞を形質移入した。24ウェルプレートのそれぞれのウェルについて、合計500ngのプラスミドを使用した。
293FT細胞を上記プラスミドDNAにより形質移入した。細胞を37℃において形質移入後72時間インキュベートしてからゲノムDNAを抽出した。ゲノムDNAは、QuickExtract DNA Extraction Solution(Epicentre)を製造業者のプロトコルに従って使用して抽出した。手短に述べると、ペレット化細胞をQuickExtract溶液中で再懸濁させ、65℃において15分間および98℃において10分間インキュベートした。それぞれの遺伝子についてのCRISPR標的部位をフランキングするゲノム領域を、PCR増幅し(表Eに列記のプライマー)、QiaQuick Spin Column(Qiagen)を製造業者のプロトコルに従って使用して産物を精製した。合計400ngの精製PCR産物を2μlの10×Taq DNA Polymerase PCR緩衝液(Enzymatics)と混合し、超純水で20μlの最終容量とし、リアニーリングプロセスに供してヘテロ二本鎖形成を可能とした:95℃において10分間、−2℃/秒における傾斜で95℃から85℃、−0.25℃/秒における85℃から25℃、および25℃において1分間維持。リアニーリング後、産物をSURVEYORヌクレアーゼおよびSURVEYORエンハンサーS(Transgenomics)により製造業者の推奨プロトコルに従って処理し、4〜20%のNovex TBEポリアクリルアミドゲル(Life Technologies)上で分析した。ゲルをSYBR Gold DNA染色(Life Technologies)により30分間染色し、Gel Docゲルイメージングシステム(Bio−rad)によりイメージングした。定量は、相対バンド強度に基づくものであった。
ヒト、マウス、ラット、ゼブラフィッシュ、ミバエ、および線虫(C.elegans)ゲノム中の化膿性連鎖球菌(S.pyogenes)SF370Cas9(SpCas9)酵素についてのユニーク標的部位を同定するため、本出願人らは、DNA配列の両方の鎖をスキャンし、考えられる全てのSpCas9標的部位を同定するためのソフトウェアパッケージを開発した。この実施例について、それぞれのSpCas9標的部位を20bp配列と、それに続くNGGプロトスペーサー隣接モチーフ(PAM)配列として操作上定義し、本出願人らは、全ての染色体上のこの5’−N20−NGG−3’定義を満たす全ての配列を同定した。非特異的ゲノム編集を防止するため、全ての潜在的な部位を同定した後、全ての標的部位をそれらが関連参照ゲノム中で出現する回数に基づきフィルタリングした。例えば、PAM配列から5’側の約11〜12bp配列であり得る「シード」配列により付与されるCas9活性の配列特異性を利用するため、5’−NNNNNNNNNN−NGG−3’配列を関連ゲノム中でユニークであると選択した。全てのゲノム配列をUCSCゲノムブラウザからダウンロードした(ヒトゲノムhg19、マウスゲノムmm9、ラットゲノムrn5、ゼブラフィッシュゲノムdanRer7、キイロショウジョウバエ(D.melanogaster)ゲノムdm4および線虫(C.elegans)ゲノムce10)。全探索結果は、UCSCゲノムブラウザ情報を使用して閲覧利用可能である。ヒトゲノム中の一部の標的部位の例示的可視化を図21に提供する。
U6−短鎖tracrRNA(化膿連鎖球菌(Streptococcus pyogenes)SF370):
本出願人らは、CRISPR関連エンドヌクレアーゼCas9を使用して肺炎連鎖球菌(Streptococcus pneumoniae)および大腸菌(Escherichia coli)のゲノム中に正確な突然変異を導入した。このアプローチは、非突然変異細胞を殺傷するためのターゲティングされる部位におけるCas9指向開裂に依存し、選択可能なマーカーまたはカウンターセレクション系の必要性を回避した。Cas9特異性は、編集テンプレート上で担持される単一および多ヌクレオチド変化を作製するように短鎖CRISPR RNA(crRNA)配列を変化させることによりリプログラミングした。2つのcrRNAの同時使用により、複数の突然変異導入が可能になった。肺炎連鎖球菌(S.pneumoniae)において、Cas9開裂から生存した細胞のほぼ100%が、所望の突然変異を含有し、大腸菌(E.coli)におけるリコンビニアリングとの組合せで使用された場合は65%が所望の突然変異を含有した。本出願人らは、もっぱら、ターゲティング可能な配列の範囲を定義するためのCas9標的要件を徹底的に分析し、それらの要件に合致しない編集部位のための方針を示し、このことは、細菌ゲノムエンジニアリングのためのこの技術の多用途性を示唆した。
肺炎連鎖球菌(S.pneumoniae)株crR6は、バクテリオファージφ8232.5中に存在する標的配列を開裂するCas9ベースCRISPR系を含有する。この標的を第2の株R68232.5のsrtA染色体遺伝子座中にインテグレートした。PAM領域中の突然変異を含有する変化標的配列を第3の株R6370.1のsrtA遺伝子座中にインテグレートし、この株をCRISPR開裂に対して「免疫性」とした(図28a)。本出願人らは、R68232.5およびR6370.1細胞をcrR6細胞からのゲノムDNAにより形質転換し、R68232.5細胞の良好な形質転換が標的遺伝子座の開裂および細胞死をもたらすはずであることを予測した。この予測とは逆に、本出願人らは、R6370.1形質転換体よりも約10倍小さい効率にもかかわらず、R68232.5形質転換体を単離した(図28b)。8つのR68232.5形質転換体の遺伝子分析(図28)により、大多数は、φ8232.5標的をCas9認識に要求されるプロトスペーサーを含有しないcrR6ゲノム野生型srtA遺伝子座により置き換えることによりCas9ターゲティングの毒性を排除する二重組換えイベントの産物であることが明らかになった。これらの結果は、ゲノム遺伝子座をターゲティングするCRISPR系(ターゲティング構築物)の、ターゲティングされる遺伝子座中への組換えのためのテンプレート(編集テンプレート)と一緒の同時導入はターゲティングされるゲノム編集をもたらす証明であった(図23a)。
ランダム化PAM:ランダム化PAM実験について、crR6について3,429,406リード、R6について3,253,998リードが得られた。これらの半数のみがPAM標的に対応する一方、他の半数がPCR産物の他の末端をシーケンシングすることが予測される。crR6リードの1,623,008およびR6リードの1,537,131は、エラーのない標的配列を担持した。これらのリードの中のそれぞれの考えられるPAMの発生率を補足ファイルに示す。PAMの機能性を推定するため、R6試料に対するcrR6試料中のその相対比率を計算し、rijklm(式中、I、j、k、l、mは、4つの考えられる塩基の1つである)で示す。以下の統計モデルを構築した:
log(rijklm)=μ+b2i+b3j+b4k+b2b3i,j+b3b4j,k+εijklm
(式中、εは、残差であり、b2は、PAMの第2の塩基の効果であり、b3は、第3の塩基の効果であり、b4は、第4の塩基の効果であり、b2b3は、第2の塩基と第3の塩基との間の相互作用であり、b3b4は、第3の塩基と第4の塩基との間の相互作用である)。分散分析を実施した。
NAGNNパターンは、全ての他のパターンと有意に異なるが、NGGNNよりもかなり少ない効果を有する(以下のテューキーのHSD検定参照)。
ランダム化標的実験について、crR6について540,726リードが、R6について753,570リードが得られた。上記のとおり、リードの半数のみがPCR産物の目的末端をシーケンシングすることが予測される。エラーフリーまたは単一の点突然変異を有する標的を担持するリードをフィルタリングした後、crR6およびR6についてそれぞれ217,656および353,141リードが残存した。R6試料に対するcrR6試料におけるそれぞれの突然変異体の相対比率を計算した(図24c)。シード配列の外側(PAMから13〜20塩基離れている)の全ての突然変異は、完全干渉を示す。これらの配列を参照として使用してシード配列の内側の他の突然変異が干渉を有意に破壊し得ると考えられるか否かを決定した。MASS Rパッケージのfitdistr関数を使用して正規分布をこれらの配列にフィットさせた。フィットされた分布の0.99分位点を図24cに点線として示す。図72は、フィットされた正規分布(黒線)および0.99分位点(点線)を有するデータ密度のヒストグラムを示す。
本出願人らは、tracrRNAおよびダイレクトリピート配列を突然変異させ、またはキメラガイドRNAを突然変異させて細胞中のRNAを向上させた。
最適化tracrRNA1(下線は突然変異):
元のガイドRNA:
本出願人らは、図4に示されるガイドキメラRNAを設計した。
CRISPR−Cas系は、細菌から古細菌にわたる多様な種により用いられる侵入外因性DNAに対する適応免疫機序である。II型CRISPR−Cas9系は、CRISPR遺伝子座中への外来DNAの「獲得」を担うタンパク質をコードする遺伝子のセット、およびDNA開裂機序の「実行」をコードする遺伝子のセットからなり;これらは、DNAヌクレアーゼ(Cas9)、非コードトランス活性化crRNA(tracrRNA)、およびダイレクトリピートによりフランキングされている外来DNA由来スペーサーのアレイ(crRNA)を含む。Cas9による成熟時、tracRNAおよびcrRNA二本鎖は、Cas9ヌクレアーゼをスペーサーガイド配列により規定される標的DNA配列にガイドし、開裂に要求され、それぞれのCRISPR−Cas系に特異的な標的DNA中の短鎖配列モチーフ付近のDNAの二本鎖切断を媒介する。II型CRISPR−Cas系は、細菌界全体にわたり見出されており、Cas9タンパク質配列およびサイズ、tracrRNAおよびcrRNAダイレクトリピート配列、それらのエレメントのゲノム構成、および標的開裂のためのモチーフ要件は高度に多様である。ある種は、複数の区別されるCRISPR−Cas系を有し得る。
細胞培養および形質移入
ヒト胚腎臓(HEK)細胞系293FT(Life Technologies)を、10%のウシ胎仔血清(HyClone)、2mMのGlutaMAX(Life Technologies)、100U/mLのペニシリン、および100μg/mLのストレプトマイシンが補給されたダルベッコ改変イーグル培地(DMEM)中で37℃において5%のCO2インキュベーションで維持した。
293FT細胞を上記のとおりプラスミドDNAにより形質移入した。細胞を37℃において形質移入後72時間インキュベートしてからゲノムDNAを抽出した。ゲノムDNAは、QuickExtract DNA Extraction Solution(Epicentre)を製造業者のプロトコルに従って使用して抽出した。手短に述べると、ペレット化細胞をQuickExtract溶液中で懸濁させ、65℃において15分間および98℃において10分間インキュベートした。
ノザンブロットを、上記のとおり実施した。手短に述べると、RNAを95℃に5分間加熱してから8%の変性ポリアクリルアミドゲル(SequaGel,National Diagnostics)上にロードした。その後、RNAを事前にハイブリダイズさせたHybond N+メンブレン(GE Healthcare)に転写し、Stratagene UV Crosslinker(Stratagene)により架橋した。プローブをT4ポリヌクレオチドキナーゼ(New England Biolabs)を用いて[ガンマ−32P]ATP(Perkin Elmer)により標識した。洗浄後、メンブレンを蛍光スクリーンに1時間曝露し、phosphorimager(Typhoon)によりスキャンした。
HEK293FT細胞を、上記のとおりCas9により形質移入した。ゲノムDNAをDNeasy Blood&Tissue Kit(Qiagen)により単離し、バイサルファイトをEZ DNA Methylation−Lightning Kit(Zymo Research)により変換した。バイサルファイトPCRは、Bisulfite Primer Seekerを使用して設計されたプライマー(Zymo Research、表JおよびK)を用いてKAPA2G Robust HotStart DNA Polymerase(KAPA Biosystems)を使用して実施した。得られたPCRアンプリコンをゲル精製し、EcoRIおよびHindIIIにより消化し、形質転換前にpUC19骨格中にライゲートした。次いで、個々のクローンをサンガーシーケンシングしてDNAメチル化状態を評価した。
HEK293FT細胞を上記のとおりCas9により形質移入した。次いで、ホールセル溶解物を、Protease Inhibitor Cocktail(Roche)が補給された溶解緩衝液(20mMのHEPES、100mMのKCl、5mMのMgCl2、1mMのDTT、5%のグリセロール、0.1%のTriton X−100)を用いて調製した。カスタムオリゴ(実施例10)およびHiScribe T7 In Vitro Transcription Kit(NEB)を製造業者の推奨プロトコルに従って使用してT7によりドライブされるsgRNAをインビトロで転写させた。メチル化標的部位を調製するため、pUC19プラスミドをM.SssIによりメチル化し、次いでNheIにより線形化した。インビトロ開裂アッセイを以下のとおり実施した:20uLの開裂反応物について、10uLの細胞溶解物を2uLの開裂緩衝液(100mMのHEPES、500mMのKCl、25mMのMgCl2、5mMのDTT、25%のグリセロール)、インビトロ転写されたRNA、300ngのpUC19プラスミドDNAとともにインキュベートした。
96ウェルプレート中でプレーティングされたHEK293FT細胞を、Cas9プラスミドDNAおよび単一ガイドRNA(sgRNA)PCRカセットにより72時間形質移入してからゲノムDNAを抽出した(図72)。それぞれの遺伝子についてのCRISPR標的部位をフランキングするゲノム領域を、融合PCR法により増幅してIllumina P5アダプターおよびユニークな試料特異的バーコードを標的アンプリコン(図73に記載の模式図)に付着させた(図74、図80(実施例10)。PCR産物は、EconoSpin96ウェルFilter Plates(Epoch Life Sciences)を製造業者の推奨プロトコルに従って使用して精製した。
MiSeqリードは、少なくとも23の平均Phredクオリティ(Qスコア)ならびにバーコードおよびアンプリコンフォワードプライマーとの完全配列マッチを要求することによりフィルタリングした。オンおよびオフターゲット遺伝子座からのリードは、標的部位の上流および下流の50ヌクレオチド(合計120bp)を含むアンプリコン配列に対してSmith−Watermanアラインメントを最初に実施することにより分析した。その一方、アラインメントを標的部位の上流の5ヌクレオチドから下流の5ヌクレオチド(合計30bp)のインデルについて分析した。これらのアラインメントの一部がMiSeqリード自体の外側に収まる場合、またはマッチした塩基対がそれらの全長の85%未満を含む場合に分析された標的領域を廃棄した。
24ウェルプレート中でプレーティングされた293FT細胞を、上記のとおり形質移入した。形質移入72時間後、トータルRNAをmiRNeasy Micro Kit(Qiagen)により回収した。sgRNAについての逆鎖合成は、qScript Flex cDNAキット(VWR)およびカスタム第1鎖合成プライマー(表JおよびK)を用いて実施した。qPCR分析は、GAPDHを内因性対照として使用してFast SYBR Green Master Mix(Life Technologies)およびカスタムプライマー(表JおよびK)を用いて実施した。相対定量をΔΔCT法により算出した。
全ての配列は、5’から3’方向である。U6転写のため、下線付きのTのストリングが転写ターミネーターとして機能する。
>U6−短鎖tracrRNA(化膿性連鎖球菌(Streptococcus pyogenes)SF370)
オリゴ相同組換え試験は、異なるCas9バリアントおよび異なるHRテンプレート(オリゴ対プラスミド)にわたる効率の比較である。
近年の大規模シーケンシング構想は、疾患に関連する多数の遺伝子を生じさせた。遺伝子の発見は、その遺伝子が何であるか、およびいかにそれが疾患表現型をもたらすのかの理解の始まりにすぎない。候補遺伝子を研究するための現在の技術およびアプローチは、緩慢で煩雑である。代表的な基準である遺伝子ターゲティングおよび遺伝子ノックアウトは、金銭および研究人材の両方の観点から時間および資源のかなりの投資を要求する。本出願人らは、hSpCas9ヌクレアーゼを利用して多くの標的遺伝子をターゲティングし、任意の他の技術と比較して高い効率および低いターンアラウンドでそれを行うように設定した。hSpCas9の高い効率のため、本出願人らは、RNAインジェクションをマウス接合子中に行い、mESCにおけるいかなる予備遺伝子ターゲティングを行うことも必要とせずにゲノム改変動物を直ちに得ることができる。
図67は、転写活性化活性を有するCRISPR−TF(転写因子)の設計を示す。キメラRNAをU6プロモーターにより発現させる一方、3つのNLSおよびVP64機能ドメインに作動可能に結合しているCas9タンパク質のヒトコドン最適化二重突然変異体バージョン(hSpCas9m)をEF1aプロモーターにより発現させる。二重突然変異D10AおよびH840Aにより、cas9タンパク質がいかなる開裂も導入し得なくなるが、キメラRNAによりガイドされた場合に標的DNAに結合するその能力は維持された。
293FT細胞を、2つの成分を含有するプラスミドにより形質移入した:(1)異なるNLS設計を有するCas9(野生型ヒトコドン最適化SpCas9)の発現をドライブするEF1aプロモーター(2)ヒトEMX1遺伝子座をターゲティングする同一キメラRNAをドライブするU6プロモーター。
Cas9を送達する方法
Chlamydomonas Resource Centerからのコナミドリムシ(Chlamydomonas reinhardtii)株CC−124およびCC−125を、エレクトロポレーションに使用する。エレクトロポレーションプロトコルは、GeneArt Chlamydomonas Engineeringキットからの標準的な推奨プロトコルに従う。
pChlamy1−Cas9:
転写を人工的に制御する技能は、遺伝子機能の研究および所望の特性を有する合成遺伝子ネットワークの構築の両方に不可欠である。本出願人らは、本明細書において、プログラマブル転写リプレッサーとしてのRNAによりガイドされるCas9タンパク質の使用を記載する。
1.1つ以上のベクターを含むベクター系であって、
a.traerメイト配列およびガイド配列をtraerメイト配列の上流に挿入するための1つ以上の挿入部位に作動可能に結合している第1の調節エレメント(ガイド配列は、発現された場合、真核細胞中の標的配列へのCRISPR複合体の配列特異的結合を指向し、CRISPR複合体は、(1)標的配列にハイブリダイズされるガイド配列、および(2)traer配列にハイブリダイズされるtraerメイト配列と複合体形成しているCRISPR酵素を含む);ならびに
b.核局在化配列を含む前記CRISPR酵素をコードする酵素コード配列に作動可能に結合している第2の調節エレメント
を含み;
成分(a)および(b)は、系の同一または異なるベクター上にある
ベクター系。
b.核局在化配列を含む前記CRISPR酵素をコードする酵素コード配列に作動可能に結合している第2の調節エレメント
を含む真核宿主細胞。
a.traerメイト配列およびガイド配列をtraerメイト配列の上流に挿入するための1つ以上の挿入部位に作動可能に結合している第1の調節エレメント(ガイド配列は、発現された場合、真核細胞中の標的配列へのCRISPR複合体の配列特異的結合を指向し、CRISPR複合体は、(1)標的配列にハイブリダイズされるガイド配列、および(2)traer配列にハイブリダイズされるtraerメイト配列と複合体形成しているCRISPR酵素を含む);ならびに/または
b.核局在化配列を含む前記CRISPR酵素をコードする酵素コード配列に作動可能に結合している第2の調節エレメント
を含むキット。
a.前記核酸配列を受容および/または保存するように構成された記憶装置;および
b.(i)前記核酸配列内のCRISPRモチーフ配列を局在化し、(ii)前記局在化されたCRISPRモチーフ配列に隣接する配列を、CRISPR複合体が結合する候補標的配列として選択するようにプログラミングされた単独または組合せにおける1つ以上のプロセッサ
を含むシステム。
a.1つ以上のベクターを真核細胞に導入すること(1つ以上のベクターは、CRISPR酵素、traerメイト配列に結合しているガイド配列、およびtraer配列の1つ以上の発現をドライブする);および
b.CRISPR複合体を標的ポリヌクレオチドに結合させて前記疾患遺伝子内の標的ポリヌクレオチドの開裂を生じさせ(CRISPR複合体は、(1)標的ポリヌクレオチド内の標的配列にハイブリダイズされるガイド配列、および(2)traer配列にハイブリダイズされるtraerメイト配列と複合体形成しているCRISPR酵素を含む)、それにより、突然変異疾患遺伝子を含むモデル真核細胞を生成することを含む方法。
a.試験化合物を、段落86〜90のいずれか1つに記載のモデル細胞と接触させること;および
b.前記疾患遺伝子中の前記突然変異に関連する細胞シグナリングイベントの低減または増大を示すリードアウトの変化を検出し、それにより、前記疾患遺伝子に関連する前記細胞シグナリングイベントをモジュレートする前記生物活性剤を開発することを含む方法。
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Claims (23)
- エンジニアリングされた、天然に存在しないCRISPR−Casベクター系であって、
a)Cas9タンパク質をコードするヌクレオチド配列に作動可能に結合している第1の調節エレメントであって、前記Cas9タンパク質が1つ以上の核局在化シグナル(NLS)を含み、1以上の異種タンパク質ドメインに融合している、第1の調節エレメント、および
b)CRISPR−Cas系キメラガイドRNAをコードするヌクレオチド配列に作動可能に結合している第2の調節エレメントであって、前記キメラガイドRNAが、真核細胞中のPAMに隣接したゲノム標的配列にハイブリダイズすることができ、CRISPR−Cas複合体の標的配列への配列特異的結合に導くことができるガイド配列、tracr配列にハイブリダイズすることができるtracrメイト配列、およびtracr配列を含み、前記tracr配列が30以上のヌクレオチド長である、第2の調節エレメント
を含む1つ以上のベクターを含み、
前記成分(a)および(b)が、前記系の同一のまたは異なるベクター上にある、系。 - 前記成分(a)および(b)が、同一のベクター上にある、請求項1に記載の系。
- 前記成分(a)および(b)が、異なるベクター上にある、請求項1に記載の系。
- 前記ベクターがウイルスベクターである、請求項1〜3のいずれか一項に記載の系。
- 前記ウイルスベクターがアデノ随伴ウイルスベクターである、請求項4に記載の系。
- エンジニアリングされた、天然に存在しないCRISPR−Cas系であって、
a)1つ以上のNLSを含み、1以上の異種タンパク質ドメインに融合しているCas9タンパク質、又は1つ以上のNLSを含み、1以上の異種タンパク質ドメインに融合しているCas9タンパク質をコードするポリヌクレオチド;
b)CRISPR−CasキメラガイドRNAであって、真核細胞中のPAMに隣接したゲノム標的配列にハイブリダイズすることができ、CRISPR−Cas複合体の標的配列への配列特異的結合に導くことができるガイド配列、tracr配列にハイブリダイズすることができるtracrメイト配列、およびtracr配列を含み、前記tracr配列が30以上のヌクレオチド長である、CRISPR−CasキメラガイドRNA:
を含む、系。 - tracr配列が、少なくとも40ヌクレオチド長である、請求項1〜6のいずれか一項に記載の系。
- tracr配列が、少なくとも50ヌクレオチド長である、請求項1〜7のいずれか一項に記載の系。
- 異種タンパク質ドメインが、以下の活性:メチラーゼ活性、デメチラーゼ活性、転写活性化活性、転写抑制活性、転写放出因子活性、ヒストン修飾活性、RNA開裂活性および核酸結合活性の1つ以上を有する、請求項1〜8のいずれか一項に記載の系。
- 異種タンパク質ドメインが、GFPである、請求項1〜8のいずれか一項に記載の系。
- 前記Cas9タンパク質が、SV40ウイルスラージT抗原のNLS(PKKKRKV)を含む、請求項1〜10のいずれか一項に記載の系。
- 前記Cas9タンパク質が、2つ以上のNLSを含む、請求項1〜11のいずれか一項に記載の系。
- 少なくとも1つのNLSが、前記Cas9タンパク質のアミノ末端またはその付近にあり、かつ/または少なくとも1つのNLSが、前記Cas9タンパク質のカルボキシ末端またはその付近にある、請求項12に記載の系。
- 少なくとも1つのNLSが、前記Cas9タンパク質のアミノ末端またはその付近にあり、かつ少なくとも1つのNLSが、前記Cas9タンパク質のカルボキシ末端またはその付近にある、請求項12に記載の系。
- 前記Cas9タンパク質が、対応する野生型Cas9タンパク質に関して変異されており、変異タンパク質が、標的ポリヌクレオチドの一方の鎖を切断する能力を欠くニッカーゼである、請求項1〜14のいずれか一項に記載の系。
- 前記Cas9タンパク質が、RuvC I、RuvC II、またはRuvC IIIの触媒ドメイン中に1つ以上の変異を含む、請求項15に記載の系。
- 前記Cas9タンパク質が、Streptococcus pyogenes Cas9(SpCas9)タンパク質の位置番号付けを基準にしてD10A、H840A、N854A、およびN863Aからなる群から選択される変異を含む、請求項15に記載の系。
- 前記Cas9タンパク質の2以上の触媒ドメインが、対応する野生型Cas9タンパク質に関して変異されており、変異タンパク質が、全てのDNA切断活性を実質的に欠いている、請求項1〜14のいずれか一項に記載の系。
- 前記Cas9タンパク質が、Streptococcus pyogenes Cas9(SpCas9)タンパク質の位置番号付けを基準にしてD10A変異と、H840A、N854AおよびN863A変異のうちの少なくとも一つとを含む、請求項18に記載の系。
- 前記Cas9をコードするヌクレオチド配列が、哺乳動物細胞またはヒト細胞での発現のためにコドン最適化されている、請求項1〜19のいずれか一項に記載の系。
- ゲノムエンジニアリングのための請求項1〜20のいずれか一項に記載の系の使用であって、ただし前記使用がヒトの生殖細胞系の遺伝子的同一性を改変するための方法を含まず、かつヒト又は動物の身体を処置するための方法でない、使用。
- 非ヒトトランスジェニック動物又はトランスジェニック植物の製造における、請求項1〜20のいずれか一項に記載の系の使用。
- エクスビボで哺乳動物細胞の1つ以上の遺伝子産物の発現を変化させるための、請求項1〜20のいずれか一項に記載の系の使用。
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