JP2015503535A - 改変されたcascadeリボ核タンパク質およびそれらの用途 - Google Patents
改変されたcascadeリボ核タンパク質およびそれらの用途 Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
Description
したがって、第1の態様では、本発明は、少なくとも以下のCRISPRタンパク質関連サブユニット:
−配列番号3のアミノ酸配列またはこれと少なくとも18%同一の配列を有するCas7(またはCOG1857)、
−配列番号4のアミノ酸配列またはこれと少なくとも17%同一の配列を有するCas5(またはCOG1688)、および
−配列番号5のアミノ酸配列またはこれと少なくとも16%同一の配列を有するCas6(またはCOG1583)を含み、
前記サブユニットの少なくとも1つが、核酸またはクロマチンを改変する活性、視覚化する活性、転写活性化する活性、または転写抑制する活性を提供するさらなるアミノ酸配列を含む、抗ウイルス防御のためのクラスター化された等間隔に間隔が置かれた短いパリンドローム反復(CRISPR)関連複合体(Cascade)、Cascadeタンパク質複合体、またはその一部を提供する。
>gi|16130667|ref|NP_417240.1|Cascade抗ウイルス複合体タンパク質を含むCRISP RNA(crRNA)[大腸菌K−12株MG1655亜株]
>gi|16130666|ref|NP_417239.1|Cascade抗ウイルス複合体タンパク質を含むCRISP RNA(crRNA)[大腸菌K−12株MG1655亜株]
>gi|16130665|ref|NP_417238.1|Cascade抗ウイルス複合体タンパク質を含むCRISP RNA(crRNA)[大腸菌K−12株MG1655亜株]
>gi|90111483|ref|NP_417237.2|Cascade抗ウイルス複合体タンパク質を含むCRISP RNA(crRNA)[大腸菌K−12株MG1655亜株]
>gi|16130663|ref|NP_417236.1|CRISPR RNA前駆体切断酵素;Cascade抗ウイルス複合体タンパク質を含むCRISP RNA(crRNA)[大腸菌K−12株MG1655亜株]
a.配列番号1のアミノ酸配列またはこれと少なくとも9%同一の配列を有するCse1サブユニット;
b.配列番号2のアミノ酸配列またはこれと少なくとも20%同一の配列を有するCse2サブユニット;
c.配列番号3のアミノ酸配列またはこれと少なくとも18%同一の配列を有するCas7サブユニット;
d.配列番号4のアミノ酸配列またはこれと少なくとも17%同一の配列を有するCas5サブユニット;
e.配列番号5のアミノ酸配列またはこれと少なくとも16%同一の配列を有するCas6サブユニットから選択され、
ここで、少なくともa、b、c、d、またはeが、核酸またはクロマチンを改変する活性、視覚化する活性、転写活性化する活性、または転写抑制する活性を有するさらなるアミノ酸配列を含む、少なくとも1つのクラスター化された等間隔に間隔が置かれた短いパリンドローム反復(CRISPR)関連タンパク質サブユニットをコードする核酸分子を提供する。
非常に高ストリンジェンシー(少なくとも90%同一の配列がハイブリッド形成可能)
ハイブリッド形成:5×SSCにて65℃で16時間
2回洗浄:2×SSCにて室温(RT)でそれぞれ15分間
2回洗浄:0.5×SSCにて65℃でそれぞれ20分間
高ストリンジェンシー(少なくとも80%同一の配列がハイブリッド形成可能)
ハイブリッド形成:5×〜6×SSCにて65℃〜70℃で16〜20時間
2回洗浄:2×SSCにてRTでそれぞれ5〜20分間
2回洗浄:1×SSCにて55℃〜70℃でそれぞれ30分間
低ストリンジェンシー(少なくとも50%同一の配列がハイブリッド形成可能)
ハイブリッド形成:6×SSCにてRT〜55℃で16〜20時間
少なくとも2回洗浄:2×〜3×SSCにてRT〜55℃でそれぞれ20〜30分間。
株、遺伝子クローニング、プラスミド、およびベクター
Cascadeを、記載のように発現および精製した(Joreら、2011)。精製を通して、20mM HEPES(pH7.5)、75mM NaCl、1mM DTT、2mM EDTAを含む緩衝液を、再懸濁および洗浄のために使用した。4mM デスチオビオチンを含む同一の緩衝液中でタンパク質溶離を行った。Cascade−Cas3融合複合体を同一の様式で発現および精製し、20mM HEPES(pH7.5)、200mM NaCl、および1mM DTTを使用して洗浄工程を行い、4mMデスチオビオチンを含む20mM HEPES(pH7.5)、75mM NaCl、1mM DTTで溶離した。
精製したCascadeまたはCascadeサブ複合体を、20mM HEPES(pH7.5)、75mM NaCl、1mM DTT、2mM EDTAを含む緩衝液中でpUC−λと混合し、37℃で15分間インキュベーションした。サンプルを0.8%TAEアガロースゲル上で一晩泳動し、SybR safe(Invitrogen)のTAEでの10000倍希釈物で30分間後染色した。BsmI(Fermentas)またはNt.BspQI(New England Biolabs)での切断を、5mM MgCl2を補足したHEPES反応緩衝液中で行った。
精製したCascadeを、20mM HEPES(pH7.5)、75mM NaCl、0.2mM DTT、0.3mM EDTAを含む緩衝液中でpUC−λ(7:1の比、250nM Cascade、35nM DNA)と混合し、37℃で15分間インキュベーションした。その後、AFMサンプル調製のために、インキュベーション混合物を、2回蒸留水で10倍希釈し、最終濃度1.2mMまでMgCl2を添加した。タンパク質−DNA複合体の沈着およびイメージングを以前に記載のように行った(Dameら,(2000)Nucleic Acids Res.28:3504−3510)。
CRISPR en cas遺伝子をコードするプラスミドを保有するBL21−AI細胞を、アンピシリン(100μg/ml)、カナマイシン(50μg/ml)、ストレプトマイシン(50μg/ml)、およびクロラムフェニコール(34μg/ml)を含むLuria−Bertaniブロス(LB)中にて37℃で一晩成長させた。一晩培養物を新鮮な抗生物質含有LBで100倍希釈し、37℃で1時間成長させた。cas遺伝子群およびCRISPRの発現を、最終濃度0.2%のL−アラビノースおよび最終濃度1mMのIPTGの添加によって1時間誘導した。感染のために、細胞を感染多重度(MOI)4でλファージと混合した。細胞をポリ−L−リジン被覆顕微鏡スライドに適用し、40倍油浸対物レンズ(開口数1.3)、励振源(514nm)としてのアルゴンレーザー、および530〜600nmでの検出を使用したAxiovert倒立顕微鏡に基づいてZeiss LSM510共焦点レーザ走査型顕微鏡を使用して分析した。全測定のためにピンホールを203μmに設定した。
カナマイシン(50μg/ml)、ストレプトマイシン(50μg/ml)、およびクロラムフェニコール(34μg/ml)を含むLBに一晩前接種材料を接種し、0.3のOD600まで成長させた。cas遺伝子群およびCRISPRの発現を、0.2%L−アラビノースおよび1mM IPTGで45分間誘導した。細胞を4℃の遠心分離によって回収し、100mM RbCl2、50mM MnCl2、30mM酢酸カリウム、10mM CaCl2、および15%グリセロール(pH5.8)を含む氷冷緩衝液での再懸濁によってコンピテントにした。インキュベーション3時間後、細胞を回収し、10mM MOPS、10mM RbCl、75mM CaCl2、15%グリセロール(pH6.8)を含む緩衝液に再懸濁した。80ngのpUC−λを添加し、その後に42℃で1分間熱ショックを与え、氷上で5分間寒冷ショックを与えることによって形質転換させた。次に、細胞をLB中にて37℃で45分間成長させ、0.2%L−アラビノース、1mM IPTG、アンピシリン(100μg/ml)、カナマイシン(50μg/ml)、ストレプトマイシン(50μg/ml)、およびクロラムフェニコール(34μg/ml)を含むLB−寒天プレート上にプレートした。
ファージ感染に対する宿主感受性を、(Brounsら(2008)Science 321,960−964)において見られるように病原性λファージ(λvir)を使用して試験した。宿主の感染感受性を、Brounsら(2008)に記載のようにプラーク形成効率(アンチ−λ CRISPRを含む株の非ターゲティングR44 CRISPRを含む株に対するプラーク数の比)として計算した。
本発明者らは、改良されたFokIヌクレアーゼをCse1のN末端に翻訳的に融合して、それぞれFokIKKR−Cse1およびFokIELD−Cse1であるCse1のバリアントを生成した。これら2つのバリアントを、Cascadeサブユニット(Cse2、Cas7、Cas5、およびCas6e)および均一なスペーサーを有する2つの異なるCRISPRプラスミドのうちの1つと同時発現させる。これにより、CascadeKKR複合体に均一のP7−crRNAが負荷され、CascadeELD複合体に均一のM13 g8−crRNAが負荷される。これらの複合体を、Jore、M.M.、ら、(2011)Nat.Struct.Mol.Biol.18(5):529−536に記載のようにN末端StrepIIタグ化Cse2を使用して精製する。さらに、N末端HISタグ化FokIを使用したさらなる精製工程を行って、全長およびインタクトなCascade−ヌクレアーゼ融合複合体を確実に精製することができる。
複合体の特異性および活性を、基質として人為的に構築した標的プラスミドを使用して試験した。このプラスミドは、両FokIドメインが相互に対面するように相反する鎖上にM13およびP7結合部位を含む(図11を参照のこと)。Cascade結合部位間の距離は、25塩基対と50塩基対との間(5bpづつ増加)で変化する。Cascadeの結合部位が4つの公知のPAM配列のいずれかと隣接する必要があるので(5’−プロトスペーサー−CTT/CAT/CTC/CCT−3’)、この距離範囲により、ほとんどの任意の所与の配列のためのかかる対をデザインするのに十分な柔軟性が得られる。
標的プラスミドの配列。数字は、M13標的部位とP7標的部位との間の距離を示す(プロトスペーサーは太字、PAMに下線)。
使用したヒトCCR5標的遺伝子選択およびCRISPRデザインは以下である。
>CRISPRアレイred1(斜体=スペーサー、太字=リピート)
Cascadeは、多サブユニットタンパク質−RNA複合体として非常に安定しており、mg単位の量にて大腸菌内で容易に産生される。大腸菌から精製されたそのインタクトな形態での複合体のトランスフェクションまたは微量注入を、送達方法として使用する(図12を参照のこと)。図12に示すように、Cascade−FokIヌクレアーゼを大腸菌から精製し、タンパク質トランスフェクション小胞中に被包する。次いで、これらをヒトHepG2細胞の細胞膜と融合して細胞質内にヌクレアーゼを放出させる(工程2)。次いで、NLS配列は、核孔通過を容易にするインポーチンタンパク質によって認識される(工程3)。次いで、CascadeKKR(白抜きの四角)およびCascadeELD(黒塗りの四角)は、その標的部位を見出して切断し(工程4)、それにより、標的部位を変化させるDNA修復経路を誘導して所望の変化を誘導するであろう。CascadeKKR/ELDヌクレアーゼは1回のみ作用することが必要であり、DNA上にコードされて細胞内に恒久的に存在する必要はない。
トランスフェクションした細胞を、数日間培養および継代する。次いで、in vivoでの標的DNA切断効率を、Guschin,D.Y.,ら(2010)Methods Mol.Biol.,649:247−256のサーベイヤーアッセイの使用によってアッセイする。簡潔に述べれば、標的DNA遺伝子座のPCRアンプリコンを、未処置細胞由来のPCRアンプリコンと1:1で混合するであろう。これらを加熱し、アニーリングさせ、NHEJによって誤って修復された標的部位でミスマッチが生じる。次いで、ミスマッチヌクレアーゼを使用して、ミスマッチしたDNA分子のみを切断し、これにより、CascadeKKR/ELDによる標的DNA切断の完了時に最大で50%切断される。次いで、この手順を、処置した細胞の標的DNAアンプリコンの配列決定によって追跡した。本アッセイにより、送達手順が迅速に評価および至適化される。
上記で説明するようにCascade−ヌクレアーゼ複合体を構築した。StrepIIタグ化Cse2サブユニットを使用した大腸菌からの親和性精製により、未変性Cascadeと比較した場合に予想される化学量論を有する複合体が得られる。図13を参照すると、これは、Streptactinのみを使用した精製24時間後の未変性Cascade(1)、P7 CrRNAを有するCascadeKKR、およびM13 CrRNAを有するCascadeELDの化学量論を示す。未変性Cascade(1)中のバンドは、上から下に以下を示す:Cse1、Cas7、Cas5、Cas6e、Cse2。CascadeKKR/ELDは、FokI−Cse1融合バンドおよびタンパク質分解の結果としてのFokIの小部分を有するCse1を示すさらなるバンドを示す。
Cascadeヌクレアーゼ融合物のデザインを、真核細胞の核内輸送を可能にするための核小体局在シグナル(NLS)が組み込まれるように改変した。この目的のために、サルウイルスSV40のラージT抗原由来のタンデムモノパータイトNLS(配列:PKKKRKVDPKKKRKV)をFokI−Cse1融合タンパク質のN末端に翻訳的に融合し、N末端にはHis6−タグが直接先行していた。His6−タグ(配列:MHHHHHH)により、StrepII精製後にさらなるNi2+−樹脂による親和性精製工程が可能である。このさらなる工程により、全長Cascade−ヌクレアーゼ融合複合体のみの単離が保証され、非産生性ヌクレアーゼ対を形成する標的部位への非インタクトなCascade複合体の結合を排除することによって切断効率が増大する。
CascadeKKR/ELDの活性および特異性を、上記のようにin vitroでアッセイした。図14Aは、CascadeKKR/ELDと37℃で30分間インキュベーションしたプロトスペーサー間の距離が25〜50bp(5bpずつ増加、レーン1〜6)であるプラスミドを示す。レーン10は、以下のその3つの可能なトポロジーでの標的プラスミドを含む:最も下のバンドはプラスミドの最初の負のスーパーコイル(nSC)形態を示し、真ん中のバンドは直鎖状化形態(XbaIによって切断)を示し、一方、上のバンドは開環(OC)形態(Nt.BbrCIでのニッキング後)を示す。レーン7は、両方の結合部位を除去したプラスミドのインキュベーションを示す(ネガティブコントロール)。したがって、図14Aは、結合部位が25〜50塩基対(5bpずつ増加)によって分離している種々の標的プラスミドを使用した典型的な切断アッセイを示す(レーン1〜6)。25〜50bpの距離を有するこれらのプラスミドを、それぞれアンチP7およびM13 crRNAを保有するCascadeKKR/ELDとインキュベーションした。結合部位を含まないプラスミドはコントロールとしての役割を果たした(レーン7)。元のプラスミドは負のスーパーコイル形態で存在し(nSC、コントロールレーン8)、ニッキングまたは直鎖状化した産物は明確に区別可能である。インキュベーションの際、結合部位が30、35、および40塩基対によって分離された場合に直鎖状切断産物が形成される(レーン2、3、4)。25、45、および50塩基対の距離で(レーン1、5、6)、標的プラスミドは不完全に切断されてニッキングされた形態(OC)が得られるようであった。これらの結果は、30bpと40bpとの間の距離を有するプラスミドにおける切断が最良であり、それにより、任意の所与の遺伝子座のためにcrRNA対をデザインする場合に十分な柔軟性が得られる。より短い距離およびより長い距離は共にニッキング活性が増加し、DSBが減少する。2つのプロトスペーサーが除去されたプラスミドはほとんど活性がなく、標的特異性を示す(レーン7)。
切断アッセイに最適な緩衝液条件を評価するためおよび複合体の活性が生理学的条件で予想されるかどうかを評価するために、以下の2つの緩衝液を選択した:(1)NEB4(New England Biolabs、50mM 酢酸カリウム、20mM Tris−酢酸塩、10mM酢酸マグネシウム、1mMジチオトレイトール、pH7.9)および(2)緩衝液O(Fermentas、50mM Tris−HCl、10mM MgCl2、100mM NaCl、0.1mg/mL BSA、pH7.5)。2つのうちで、市販のインタクトなFokI酵素の最適な活性にはNEB4が推奨される。良好な活性および特異性を得るためにクイックスクリーンから緩衝液Oを選択した(データ示さず)。図14Bは、異なる緩衝液および異なるインキュベーション時間を使用したインキュベーションを示す。レーン1〜4は、Fermentas緩衝液Oを使用してインキュベーションし(レーン1、2は15分間、レーン3、4は30分間)、レーン5、6はNEB4とインキュベーションした(30分間)。レーン1、3、5は35bp間隔の標的プラスミドを使用し、レーン2、4、6は非標的プラスミド(結合部位なし)を使用した。レーン7、8は、CascadeKKRまたはCascadeELDのみをそれぞれ使用してインキュベーションした(緩衝液O)。レーン9は(A)と同様のトポロジーマーカーである。レーン10および11は、Cascadeを添加せずにインキュベーションした標的プラスミドおよび非標的プラスミドを示す。したがって、図14Bでは、35塩基対の距離を有する標的プラスミド(レーン1、3、5)および非標的コントロールプラスミド(レーン2、4、6)についての活性を試験した。NEB4において大量の非特異的ニッキングおよびより少ない切断が認められ(レーン5,6)、一方で緩衝液Oは大量の特異的切断およびわずかなニッキングを有する標的プラスミドにおける活性のみを示す(レーン1〜4)。この差異は緩衝液O中のNaCl濃度に原因する可能性が高く、イオン強度が高いほどタンパク質間相互作用が弱くなり、非特異的活性が少なくなる。15分間または30分間のインキュベーションは、標的プラスミドおよび非標的プラスミドの両方でほとんど差異がない(それぞれ、レーン1,2、または3,4)。予想通り、1つのCascade型のみ(P7KKRまたはM13ELD)の添加によって切断活性は得られない(レーン7、8)。この実験は、NaCl濃度が少なくとも100mM(細胞内の生理食塩水濃度(137mM NaCl)に近い)である場合にデザインされた対による特異的Cascadeヌクレアーゼ活性が生じることを示す。Cascadeヌクレアーゼ対はin vivo(真核細胞内)で完全な活性を示すことが予想される一方で、無視できるオフターゲット切断活性を示す。
35bpの間隔を有する標的プラスミド(pTarget35)中の切断部位を決定した。図15は、どのようにして配列決定によって35塩基対間隔を有する標的プラスミド中のCascadeKKR/ELDによる上流および下流の切断部位が明らかになるかを示す。図15Aは、注釈付きの潜在的切断部位を有するpTarget35内の標的領域を示す。プロトスペーサー部分を赤色および青色で示す。B)バーチャートは、配列決定したクローンにおける4つの異なる切断パターンおよびこれらの相対存在量を示す。青色バーは生成されたオーバーハングを示し、一方で、各バーの左または右の境界は左または右の切断部位を示す(注釈付けについてはBを参照のこと)。
CascadeKKR/ELDヌクレアーゼを、N末端His6−タグおよびその後に二重モノパータイト核小体局在シグナルを含むように首尾よく改変した。これらの改変されたCascadeヌクレアーゼ融合タンパク質を、2つの合成的に構築されたCRISPRアレイ(それぞれヒトCCR5遺伝子中の結合部位をターゲティングする)のうちのいずれか1つと同時発現させた。第1に、この新規のヌクレアーゼ対の活性を、このCCR5遺伝子領域を含むプラスミドの活性を試験することによってin vitroで確認する。ヌクレアーゼ対を、ヒト細胞株(例えば、HeLa細胞株)にトランスフェクションする。標的の切断効率を、上記のSurveyorアッセイを使用して評価する。
Claims (46)
- 少なくとも以下のCRISPR関連タンパク質サブユニット:
−配列番号3のアミノ酸配列またはこれと少なくとも18%同一の配列を有するCas7、
−配列番号4のアミノ酸配列またはこれと少なくとも17%同一の配列を有するCas5、および
−配列番号5のアミノ酸配列またはこれと少なくとも16%同一の配列を有するCas6を含み、
該サブユニットの少なくとも1つが、核酸またはクロマチンを改変する活性、視覚化する活性、転写活性化する活性、または転写抑制する活性を提供するさらなるアミノ酸配列を含む、
抗ウイルス防御のための、クラスター化された等間隔に間隔が置かれた短いパリンドローム反復(CRISPR)関連複合体(Cascade)、Cascadeタンパク質複合体、またはその一部。 - 前記Cas6サブユニットが、配列番号17のアミノ酸配列またはこれと少なくとも16%同一の配列を有するCas6eサブユニットである、請求項1に記載のタンパク質複合体。
- 配列番号2のアミノ酸配列またはこれと少なくとも20%同一の配列を有するCse2サブユニットをさらに含み、必要に応じて、さらなるアミノ酸配列を含むのはCse2サブユニットである、請求項1または請求項2に記載のタンパク質複合体。
- 配列番号1のアミノ酸配列またはこれと少なくとも9%同一の配列を有するCse1サブユニットをさらに含み、必要に応じて、さらなるアミノ酸配列を含むのはCse1サブユニットである、請求項1〜3のいずれか1項に記載のタンパク質複合体。
- I型CRISPR−Casシステムタンパク質複合体、好ましくはI−E亜型CRISPR−Casタンパク質複合体である、請求項4に記載のタンパク質複合体。
- 前記さらなるアミノ酸配列が、前記少なくとも1つのサブユニットに翻訳的に融合するかまたは共有結合しており、好ましくは、前記さらなるアミノ酸配列が、Cse1、Cse2、Cas7、Cas5、Cas6、またはCas6eサブユニットのうちの少なくとも1つの少なくともN末端および/またはC末端に融合または連結している、請求項1〜5のいずれか1項に記載のタンパク質複合体。
- 前記さらなるアミノ酸配列がCse1、Cse2、またはCas5サブユニットのN末端またはC末端、好ましくは、Cse1サブユニットのN末端、Cse2サブユニットのN末端、またはCas7サブユニットのN末端に融合または連結している、請求項6に記載のタンパク質複合体。
- 前記さらなるアミノ酸配列がタンパク質であり、該タンパク質が、ヘリカーゼ、ヌクレアーゼ、ヌクレアーゼ−ヘリカーゼ(例えば、Cas3)、DNAメチルトランスフェラーゼ(例えば、Dam)、またはDNAデメチラーゼ、ヒストンメチルトランスフェラーゼ、ヒストンデメチラーゼ、アセチラーゼ、デアセチラーゼ、ホスファターゼ、キナーゼ、転写(コ)アクチベーター、RNAポリメラーゼサブユニット、転写リプレッサー、DNA結合タンパク質、DNA構造化タンパク質、マーカータンパク質、レポータータンパク質、蛍光タンパク質、リガンド結合タンパク質(例えば、mCherryまたは重金属結合タンパク質)、シグナルペプチド(例えば、Tat−シグナル配列)、細胞内局在配列(例えば、核局在配列)、または抗体エピトープから必要に応じて選択される、請求項7に記載のタンパク質複合体。
- 前記ヌクレアーゼが、II型制限エンドヌクレアーゼ、好ましくはFokI、より好ましくは改変FokI(例えば、KKR SharkeyまたはELD Sharkey)から選択される、請求項8に記載のタンパク質複合体。
- 標的核酸配列と少なくとも50%同一のリボヌクレオチド配列を含むRNA分子をさらに含む前記いずれかの請求項に記載のタンパク質複合体であって、該タンパク質複合体および該RNA分子がリボ核タンパク質複合体を形成する、タンパク質複合体。
- 前記RNA分子の一部が前記標的配列と少なくとも50%同一である、請求項10に記載のリボ核タンパク質複合体。
- 前記RNA分子の前記一部がその長さに沿って前記標的配列と少なくとも実質的に相補的である、請求項11に記載のリボ核タンパク質複合体。
- 前記RNA分子の長さが35〜75残基の範囲である、請求項10〜12のいずれか1項に記載のリボ核タンパク質複合体。
- 所望の核酸配列をターゲティングするために使用した前記RNA分子の前記一部が32または33残基長である、請求項10〜13のいずれか1項に記載のリボ核タンパク質複合体。
- 前記RNA分子が前記標的配列と少なくとも実質的な相補性を有するRNA配列の5’側にある8残基を含む、請求項10〜14のいずれか1項に記載のリボ核タンパク質複合体。
- 前記RNA分子が、前記標的配列に少なくとも実質的な相補性を有する前記RNA配列の3’側にヘアピンおよびテトラヌクレオチドループ形成配列を有する、請求項10〜15のいずれか1項に記載のリボ核タンパク質複合体。
- a.配列番号1のアミノ酸配列またはこれと少なくとも9%同一の配列を有するCse1サブユニット;
b.配列番号2のアミノ酸配列またはこれと少なくとも20%同一の配列を有するCse2サブユニット;
c.配列番号3のアミノ酸配列またはこれと少なくとも18%同一の配列を有するCas7サブユニット;
d.配列番号4のアミノ酸配列またはこれと少なくとも17%同一の配列を有するCas5サブユニット;
e.配列番号5のアミノ酸配列またはこれと少なくとも16%同一の配列を有するCas6サブユニットから選択され、
ここで、少なくともa、b、c、d、またはeが、核酸またはクロマチンを改変する活性、視覚化する活性、転写活性化する活性、または転写抑制する活性を提供するさらなるアミノ酸配列を含む、少なくとも1つのクラスター化された等間隔に間隔が置かれた短いパリンドローム反復(CRISPR)関連タンパク質サブユニットをコードする単離核酸分子。 - 前記核酸またはクロマチンを改変する活性、視覚化する活性、転写活性化する活性、または転写抑制する活性を提供するさらなるアミノ酸配列が前記CRISPR関連タンパク質サブユニットに融合している、請求項17に記載の単離核酸分子。
- 前記さらなるアミノ酸配列が、ヘリカーゼ、ヌクレアーゼ、ヌクレアーゼ−ヘリカーゼ(例えば、Cas3)、DNAメチルトランスフェラーゼ(例えば、Dam)、DNAデメチラーゼ、ヒストンメチルトランスフェラーゼ、ヒストンデメチラーゼ、アセチラーゼ、デアセチラーゼ、ホスファターゼ、キナーゼ、転写(コ)アクチベーター、RNAポリメラーゼサブユニット、転写リプレッサー、DNA結合タンパク質、DNA構造化タンパク質、マーカータンパク質、レポータータンパク質、蛍光タンパク質、リガンド結合タンパク質(例えば、mCherryまたは重金属結合タンパク質)、シグナルペプチド(例えば、Tat−シグナル配列)、細胞内局在配列(例えば、核局在配列)、または抗体エピトープから選択される、請求項18に記載の単離核酸分子。
- 請求項17〜19のいずれか1項に記載の核酸分子を含む発現ベクター。
- 請求項10〜16のいずれか1項に定義のRNA分子をコードするヌクレオチド配列をさらに含む、請求項20に記載の発現ベクター。
- 標的核酸の改変、視覚化、転写活性化、または転写抑制の方法であって、該核酸を、
a.請求項10〜16のいずれか1項に記載のリボ核タンパク質複合体、または
b.請求項1〜9のいずれか1項に記載のタンパク質複合体および請求項10〜16のいずれか1項に定義のRNA分子
と接触させる工程を含む、方法。 - 細胞内の標的核酸を改変するか、視覚化するか、転写活性化するか、または転写抑制する方法であって、該細胞を請求項22に記載の発現ベクターおよび請求項10〜18のいずれか1項に定義のRNA分子をコードするヌクレオチド配列を含むさらなる発現ベクターでトランスフェクションするか、形質転換するか、または形質導入する工程を含む、方法。
- 細胞内の標的核酸を改変するか、視覚化するか、転写活性化するか、または転写抑制する方法であって、該細胞を請求項22に記載の発現ベクターでトランスフェクションするか、形質転換するか、または形質導入し、次いで、請求項10〜18のいずれか1項に定義のRNA分子を該細胞へまたは該細胞内に投与する工程を含む、方法。
- 細胞内の標的核酸を改変するか、視覚化するか、転写活性化するか、または転写抑制する方法であって、該細胞を請求項21に記載の発現ベクターでトランスフェクションするか、形質転換するか、または形質導入する工程を含む、方法。
- 前記核酸またはクロマチンを改変または視覚化する活性を有するさらなるアミノ酸配列がマーカーであり、前記マーカーが前記標的核酸またはクロマチンと会合し、好ましくは、前記マーカーがタンパク質であり、該タンパク質は必要に応じて蛍光タンパク質(例えば、緑色蛍光タンパク質(GFP)または黄色蛍光タンパク質(YFP))である、請求項22〜25のいずれか1項に記載の標的核酸を改変するかまたは視覚化する方法。
- 前記標的核酸がDNA、好ましくはdsDNAである、請求項22〜26のいずれか1項に記載の方法。
- 前記標的核酸がRNA、好ましくはmRNAである、請求項22〜26のいずれか1項に記載の方法。
- 前記核酸がdsDNAであり、前記核酸またはクロマチンを改変する活性を有するさらなるアミノ酸配列がヌクレアーゼまたはヌクレアーゼ−ヘリカーゼであり、前記改変が所望の遺伝子座での一本鎖切断または二本鎖切断である、請求項22〜26のいずれか1項に記載の標的核酸を改変する方法。
- dsDNA分子からヌクレオチド配列の少なくとも一部を除去するため(必要に応じて、遺伝子の機能をノックアウトするため)の所望の遺伝子座での細胞内のdsDNA分子の非相同末端結合方法であって、請求項29に記載の標的核酸を改変する方法を使用して二本鎖切断をつくる工程を含む、方法。
- 既存のヌクレオチド配列を改変するかまたは所望のヌクレオチド配列を挿入するための所望の遺伝子座での細胞内のdsDNA分子中への核酸の相同組換え方法であって、請求項29に記載の標的核酸を改変する方法を使用して所望の遺伝子座で一本鎖または二本鎖切断をつくる工程を含む、方法。
- 請求項22〜25のいずれか1項に記載の方法で標的核酸配列を改変する工程を含む生物における遺伝子発現を改変するか、活性化するか、または抑制する方法であって、前記核酸がdsDNAであり、前記核酸またはクロマチンを改変する活性、転写活性化する活性、または転写抑制する活性を有するさらなるアミノ酸配列が、DNA改変酵素(例えば、デメチラーゼまたはデアセチラーゼ)、転写アクチベーター、または転写リプレッサーから選択される、方法。
- 請求項22〜25のいずれか1項に記載の方法に記載のとおりに標的核酸配列を改変する工程を含む生物における遺伝子発現を改変するか、活性化するか、または抑制する方法であって、前記核酸がmRNAであり、前記核酸またはクロマチンを改変する活性、または転写活性化する活性、または転写抑制する活性を有するさらなるアミノ酸配列が、リボヌクレアーゼであり、該リボヌクレアーゼが、エンドヌクレアーゼ、3’エキソヌクレアーゼ、または5’エキソヌクレアーゼから必要に応じて選択される、方法。
- 前記細胞が原核生物である、請求項22〜33のいずれか1項に記載の方法。
- 前記細胞が、真核細胞であり、例えば、植物細胞、酵母細胞、真菌細胞、昆虫細胞、哺乳動物細胞、またはヒト細胞である、請求項22〜33のいずれか1項に記載の方法。
- 前記哺乳動物またはヒトの細胞がヒト胚性幹細胞以外の幹細胞、好ましくは単離幹細胞である、請求項35に記載の方法。
- 前記細胞をin vitroでトランスフェクションする、請求項33〜35のいずれか1項に記載の方法。
- 前記標的核酸が三次構造を有し、該三次構造が必要に応じてスーパーコイルであり、好ましくは、前記標的核酸が負のスーパーコイルである、請求項22〜36のいずれか1項に記載の方法。
- 請求項1〜9のいずれか1項に記載のCascadeタンパク質複合体を含む薬学的組成物。
- 請求項10〜16のいずれか1項に記載のリボ核タンパク質複合体を含む薬学的組成物。
- 請求項17〜19のいずれか1項に記載の単離核酸または請求項20もしくは請求項21に記載の発現ベクターを含む薬学的組成物。
- 請求項1〜9のいずれか1項に記載のCascadeタンパク質複合体および請求項10〜16のいずれか1項に定義のRNA分子を含むキット。
- 医薬として使用するための請求項1〜9のいずれか1項に記載のCascadeタンパク質複合体。
- 医薬として使用するための請求項10〜16のいずれか1項に記載のリボ核タンパク質複合体。
- 医薬として使用するための請求項17〜19のいずれか1項に記載の単離核酸。
- 医薬として使用するための請求項20または請求項21に記載の発現ベクター。
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Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20140115335A (ko) * | 2011-12-30 | 2014-09-30 | 바게닝겐 유니버시테이트 | 변형 캐스케이드 리보핵단백질 및 이의 용도 |
WO2017043573A1 (ja) * | 2015-09-09 | 2017-03-16 | 国立大学法人神戸大学 | 標的化したdna配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体 |
WO2018225858A1 (ja) * | 2017-06-08 | 2018-12-13 | 国立大学法人大阪大学 | Dnaが編集された真核細胞を製造する方法、および当該方法に用いられるキット |
WO2019039417A1 (ja) | 2017-08-21 | 2019-02-28 | 国立大学法人徳島大学 | ヌクレオチド標的認識を利用した標的配列特異的改変技術 |
US10655123B2 (en) | 2014-03-05 | 2020-05-19 | National University Corporation Kobe University | Genomic sequence modification method for specifically converting nucleic acid bases of targeted DNA sequence, and molecular complex for use in same |
US10767173B2 (en) | 2015-09-09 | 2020-09-08 | National University Corporation Kobe University | Method for converting genome sequence of gram-positive bacterium by specifically converting nucleic acid base of targeted DNA sequence, and molecular complex used in same |
WO2021149829A1 (ja) * | 2020-01-24 | 2021-07-29 | C4U株式会社 | 試料中の特定のdnaを検出する方法 |
JP2021520844A (ja) * | 2018-06-13 | 2021-08-26 | カリブー・バイオサイエンシーズ・インコーポレイテッド | 操作されたカスケード構成要素およびカスケード複合体 |
US11220693B2 (en) | 2015-11-27 | 2022-01-11 | National University Corporation Kobe University | Method for converting monocot plant genome sequence in which nucleic acid base in targeted DNA sequence is specifically converted, and molecular complex used therein |
JP2022518643A (ja) * | 2018-04-03 | 2022-03-16 | ジーフラス ライフ サイエンシズ,インク. | 配列特異的なインビボ細胞標的化 |
Families Citing this family (196)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7790756B2 (en) | 2006-10-11 | 2010-09-07 | Deciphera Pharmaceuticals, Llc | Kinase inhibitors useful for the treatment of myleoproliferative diseases and other proliferative diseases |
SG185481A1 (en) | 2010-05-10 | 2012-12-28 | Univ California | Endoribonuclease compositions and methods of use thereof |
WO2013066438A2 (en) | 2011-07-22 | 2013-05-10 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
IN2014MN00974A (ja) | 2011-12-16 | 2015-04-24 | Targetgene Biotechnologies Ltd | |
US11021737B2 (en) | 2011-12-22 | 2021-06-01 | President And Fellows Of Harvard College | Compositions and methods for analyte detection |
EP2841572B1 (en) | 2012-04-27 | 2019-06-19 | Duke University | Genetic correction of mutated genes |
LT3401400T (lt) | 2012-05-25 | 2019-06-10 | The Regents Of The University Of California | Būdai ir kompozicijos, skirtos rnr molekulės nukreipiamai tikslinės dnr modifikacijai ir rnr molekulės nukreipiamam transkripcijos moduliavimui |
DE202013012597U1 (de) | 2012-10-23 | 2017-11-21 | Toolgen, Inc. | Zusammensetzung zum Spalten einer Ziel-DNA, umfassend eine für die Ziel-DNA spezifische guide-RNA und eine Cas-Protein-codierende Nukleinsäure oder ein Cas-Protein, sowie deren Verwendung |
EP3138911B1 (en) | 2012-12-06 | 2018-12-05 | Sigma Aldrich Co. LLC | Crispr-based genome modification and regulation |
US8697359B1 (en) | 2012-12-12 | 2014-04-15 | The Broad Institute, Inc. | CRISPR-Cas systems and methods for altering expression of gene products |
PT2921557T (pt) | 2012-12-12 | 2016-10-19 | Massachusetts Inst Technology | Engenharia de sistemas, métodos e composições guia otimizadas para a manipulação de sequências |
ES2576128T3 (es) | 2012-12-12 | 2016-07-05 | The Broad Institute, Inc. | Modificación por tecnología genética y optimización de sistemas, métodos y composiciones para la manipulación de secuencias con dominios funcionales |
WO2014093694A1 (en) | 2012-12-12 | 2014-06-19 | The Broad Institute, Inc. | Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes |
DK2898075T3 (en) | 2012-12-12 | 2016-06-27 | Broad Inst Inc | CONSTRUCTION AND OPTIMIZATION OF IMPROVED SYSTEMS, PROCEDURES AND ENZYME COMPOSITIONS FOR SEQUENCE MANIPULATION |
ES2658401T3 (es) | 2012-12-12 | 2018-03-09 | The Broad Institute, Inc. | Suministro, modificación y optimización de sistemas, métodos y composiciones para la manipulación de secuencias y aplicaciones terapéuticas |
EP4234696A3 (en) | 2012-12-12 | 2023-09-06 | The Broad Institute Inc. | Crispr-cas component systems, methods and compositions for sequence manipulation |
EP2931892B1 (en) | 2012-12-12 | 2018-09-12 | The Broad Institute, Inc. | Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof |
CA3081054A1 (en) | 2012-12-17 | 2014-06-26 | President And Fellows Of Harvard College | Rna-guided human genome engineering |
US10138509B2 (en) | 2013-03-12 | 2018-11-27 | President And Fellows Of Harvard College | Method for generating a three-dimensional nucleic acid containing matrix |
KR101780885B1 (ko) | 2013-03-14 | 2017-10-11 | 카리부 바이오사이언시스 인코포레이티드 | 핵산-표적화 핵산의 조성물 및 방법 |
US10760064B2 (en) | 2013-03-15 | 2020-09-01 | The General Hospital Corporation | RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci |
CA2906553C (en) | 2013-03-15 | 2022-08-02 | The General Hospital Corporation | Rna-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci |
US9902973B2 (en) | 2013-04-11 | 2018-02-27 | Caribou Biosciences, Inc. | Methods of modifying a target nucleic acid with an argonaute |
US20140356956A1 (en) | 2013-06-04 | 2014-12-04 | President And Fellows Of Harvard College | RNA-Guided Transcriptional Regulation |
KR20160030187A (ko) | 2013-06-17 | 2016-03-16 | 더 브로드 인스티튜트, 인코퍼레이티드 | 간의 표적화 및 치료를 위한 CRISPRCas 시스템, 벡터 및 조성물의 전달 및 용도 |
WO2014204725A1 (en) | 2013-06-17 | 2014-12-24 | The Broad Institute Inc. | Optimized crispr-cas double nickase systems, methods and compositions for sequence manipulation |
WO2014204727A1 (en) | 2013-06-17 | 2014-12-24 | The Broad Institute Inc. | Functional genomics using crispr-cas systems, compositions methods, screens and applications thereof |
ES2777217T3 (es) | 2013-06-17 | 2020-08-04 | Broad Inst Inc | Suministro, modificación y optimización de sistemas de guía en tándem, métodos y composiciones para la manipulación de secuencias |
EP3011034B1 (en) | 2013-06-17 | 2019-08-07 | The Broad Institute, Inc. | Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using viral components |
US10011850B2 (en) | 2013-06-21 | 2018-07-03 | The General Hospital Corporation | Using RNA-guided FokI Nucleases (RFNs) to increase specificity for RNA-Guided Genome Editing |
CN105517579B (zh) | 2013-07-10 | 2019-11-15 | 哈佛大学校长及研究员协会 | 用于RNA向导的基因调节和编辑的正交Cas9蛋白 |
US11060083B2 (en) | 2013-07-19 | 2021-07-13 | Larix Bioscience Llc | Methods and compositions for producing double allele knock outs |
CN105392885B (zh) * | 2013-07-19 | 2020-11-03 | 赖瑞克斯生物科技公司 | 用于产生双等位基因敲除的方法和组合物 |
US11306328B2 (en) * | 2013-07-26 | 2022-04-19 | President And Fellows Of Harvard College | Genome engineering |
CN103388006B (zh) * | 2013-07-26 | 2015-10-28 | 华东师范大学 | 一种基因定点突变的构建方法 |
US9163284B2 (en) | 2013-08-09 | 2015-10-20 | President And Fellows Of Harvard College | Methods for identifying a target site of a Cas9 nuclease |
WO2015021426A1 (en) * | 2013-08-09 | 2015-02-12 | Sage Labs, Inc. | A crispr/cas system-based novel fusion protein and its application in genome editing |
US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
CA3109801C (en) | 2013-08-22 | 2024-01-09 | Andrew Cigan | Plant genome modification using guide rna/cas endonuclease systems and methods of use |
EA037850B1 (ru) | 2013-08-29 | 2021-05-27 | Тэмпл Юниверсити Оф Зе Коммонвэлс Систем Оф Хайе Эдьюкейшн | Способы и композиции для рнк-направленного лечения вич-инфекции |
US9228207B2 (en) | 2013-09-06 | 2016-01-05 | President And Fellows Of Harvard College | Switchable gRNAs comprising aptamers |
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US9322037B2 (en) | 2013-09-06 | 2016-04-26 | President And Fellows Of Harvard College | Cas9-FokI fusion proteins and uses thereof |
ES2844174T3 (es) | 2013-09-18 | 2021-07-21 | Kymab Ltd | Métodos, células y organismos |
WO2015065964A1 (en) | 2013-10-28 | 2015-05-07 | The Broad Institute Inc. | Functional genomics using crispr-cas systems, compositions, methods, screens and applications thereof |
US10584358B2 (en) | 2013-10-30 | 2020-03-10 | North Carolina State University | Compositions and methods related to a type-II CRISPR-Cas system in Lactobacillus buchneri |
LT3066201T (lt) | 2013-11-07 | 2018-08-10 | Editas Medicine, Inc. | Su crispr susiję būdai ir kompozicijos su valdančiomis grnr |
US9074199B1 (en) | 2013-11-19 | 2015-07-07 | President And Fellows Of Harvard College | Mutant Cas9 proteins |
US10787684B2 (en) | 2013-11-19 | 2020-09-29 | President And Fellows Of Harvard College | Large gene excision and insertion |
US20150165054A1 (en) | 2013-12-12 | 2015-06-18 | President And Fellows Of Harvard College | Methods for correcting caspase-9 point mutations |
KR20160089527A (ko) | 2013-12-12 | 2016-07-27 | 더 브로드 인스티튜트, 인코퍼레이티드 | 게놈 편집을 위한 crispr-cas 시스템 및 조성물의 전달, 용도 및 치료적 응용 |
WO2015089486A2 (en) | 2013-12-12 | 2015-06-18 | The Broad Institute Inc. | Systems, methods and compositions for sequence manipulation with optimized functional crispr-cas systems |
WO2015089364A1 (en) | 2013-12-12 | 2015-06-18 | The Broad Institute Inc. | Crystal structure of a crispr-cas system, and uses thereof |
KR20160097327A (ko) | 2013-12-12 | 2016-08-17 | 더 브로드 인스티튜트, 인코퍼레이티드 | 유전자 산물, 구조 정보 및 유도성 모듈형 cas 효소의 발현의 변경을 위한 crispr-cas 시스템 및 방법 |
WO2015089354A1 (en) | 2013-12-12 | 2015-06-18 | The Broad Institute Inc. | Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders |
JP6652489B2 (ja) | 2013-12-19 | 2020-02-26 | アミリス, インコーポレイテッド | ゲノム組込みのための方法 |
US10787654B2 (en) | 2014-01-24 | 2020-09-29 | North Carolina State University | Methods and compositions for sequence guiding Cas9 targeting |
EP3957735A1 (en) | 2014-03-05 | 2022-02-23 | Editas Medicine, Inc. | Crispr/cas-related methods and compositions for treating usher syndrome and retinitis pigmentosa |
EP3553176A1 (en) | 2014-03-10 | 2019-10-16 | Editas Medicine, Inc. | Crispr/cas-related methods and compositions for treating leber's congenital amaurosis 10 (lca10) |
US11339437B2 (en) | 2014-03-10 | 2022-05-24 | Editas Medicine, Inc. | Compositions and methods for treating CEP290-associated disease |
US11141493B2 (en) | 2014-03-10 | 2021-10-12 | Editas Medicine, Inc. | Compositions and methods for treating CEP290-associated disease |
EP3122880B1 (en) | 2014-03-26 | 2021-05-05 | Editas Medicine, Inc. | Crispr/cas-related methods and compositions for treating sickle cell disease |
AU2015237746B2 (en) * | 2014-03-28 | 2020-01-30 | Aposense Ltd. | Compounds and methods for trans-membrane delivery of molecules |
US11318206B2 (en) | 2014-03-28 | 2022-05-03 | Aposense Ltd | Compounds and methods for trans-membrane delivery of molecules |
WO2015155686A2 (en) * | 2014-04-08 | 2015-10-15 | North Carolina State University | Methods and compositions for rna-directed repression of transcription using crispr-associated genes |
IL286474B2 (en) | 2014-06-23 | 2023-11-01 | Massachusetts Gen Hospital | Genome-wide random identification of DSBS assessed by sequencing (guide-sequence) |
AU2015288157A1 (en) | 2014-07-11 | 2017-01-19 | E. I. Du Pont De Nemours And Company | Compositions and methods for producing plants resistant to glyphosate herbicide |
US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
AU2015308910B2 (en) | 2014-08-27 | 2017-12-07 | Caribou Biosciences, Inc. | Methods for increasing Cas9-mediated engineering efficiency |
EP3186375A4 (en) | 2014-08-28 | 2019-03-13 | North Carolina State University | NEW CAS9 PROTEINS AND GUIDING ELEMENTS FOR DNA TARGETING AND THE GENOME EDITION |
CN108064129A (zh) | 2014-09-12 | 2018-05-22 | 纳幕尔杜邦公司 | 玉米和大豆中复合性状基因座的位点特异性整合位点的产生和使用方法 |
CA2963820A1 (en) | 2014-11-07 | 2016-05-12 | Editas Medicine, Inc. | Methods for improving crispr/cas-mediated genome-editing |
KR20240013283A (ko) | 2014-12-03 | 2024-01-30 | 애질런트 테크놀로지스, 인크. | 화학적 변형을 갖는 가이드 rna |
KR102656470B1 (ko) | 2014-12-10 | 2024-04-09 | 리전츠 오브 더 유니버스티 오브 미네소타 | 질환을 치료하기 위한 유전적으로 변형된 세포, 조직 및 장기 |
EP3985115A1 (en) | 2014-12-12 | 2022-04-20 | The Broad Institute, Inc. | Protected guide rnas (pgrnas) |
KR102319192B1 (ko) | 2015-01-28 | 2021-10-28 | 카리부 바이오사이언시스 인코포레이티드 | Crispr 하이브리드 dna/rna 폴리뉴클레오티드 및 사용 방법 |
EP3265559B1 (en) | 2015-03-03 | 2021-01-06 | The General Hospital Corporation | Engineered crispr-cas9 nucleases with altered pam specificity |
WO2016153305A1 (ko) * | 2015-03-26 | 2016-09-29 | 한국생명공학연구원 | 표적 유전자 특이적 핵산 프로브 및 Fok Ι 제한효소 이량체를 이용하여 세포 내에서 표적 유전자를 특이적으로 편집하기 위한 조성물 및 이의 용도 |
CN107567499A (zh) | 2015-03-27 | 2018-01-09 | 纳幕尔杜邦公司 | 大豆u6核小rna基因启动子及其在植物小rna基因的组成型表达中的用途 |
JP6873911B2 (ja) | 2015-04-06 | 2021-05-19 | ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー | 初代細胞において標的核酸の遺伝子調節を誘導するためにインビトロで行う方法 |
JP2018522249A (ja) | 2015-04-24 | 2018-08-09 | エディタス・メディシン、インコーポレイテッド | Cas9分子/ガイドrna分子複合体の評価 |
JP7030522B2 (ja) | 2015-05-11 | 2022-03-07 | エディタス・メディシン、インコーポレイテッド | 幹細胞における遺伝子編集のための最適化crispr/cas9システムおよび方法 |
KR102451796B1 (ko) | 2015-05-29 | 2022-10-06 | 노쓰 캐롤라이나 스테이트 유니버시티 | 크리스퍼 핵산을 사용하여 박테리아, 고세균, 조류 및 효모를 스크리닝하는 방법 |
EP3302053B1 (en) | 2015-06-02 | 2021-03-17 | Monsanto Technology LLC | Compositions and methods for delivery of a polynucleotide into a plant |
CN108368502B (zh) | 2015-06-03 | 2022-03-18 | 内布拉斯加大学董事委员会 | 使用单链dna的dna编辑 |
WO2016201047A1 (en) | 2015-06-09 | 2016-12-15 | Editas Medicine, Inc. | Crispr/cas-related methods and compositions for improving transplantation |
KR20220158846A (ko) | 2015-06-15 | 2022-12-01 | 노쓰 캐롤라이나 스테이트 유니버시티 | 핵산 및 rna-기반 항미생물제를 효율적으로 전달하기 위한 방법 및 조성물 |
AU2016280893B2 (en) | 2015-06-18 | 2021-12-02 | Massachusetts Institute Of Technology | CRISPR enzyme mutations reducing off-target effects |
WO2016205759A1 (en) | 2015-06-18 | 2016-12-22 | The Broad Institute Inc. | Engineering and optimization of systems, methods, enzymes and guide scaffolds of cas9 orthologs and variants for sequence manipulation |
EP3325668B1 (en) * | 2015-07-23 | 2021-01-06 | Mayo Foundation for Medical Education and Research | Editing mitochondrial dna |
IL295616A (en) | 2015-07-31 | 2022-10-01 | Us Health | Adapted cells and treatment methods |
US9580727B1 (en) | 2015-08-07 | 2017-02-28 | Caribou Biosciences, Inc. | Compositions and methods of engineered CRISPR-Cas9 systems using split-nexus Cas9-associated polynucleotides |
JP6799586B2 (ja) | 2015-08-28 | 2020-12-16 | ザ ジェネラル ホスピタル コーポレイション | 遺伝子操作CRISPR−Cas9ヌクレアーゼ |
US9512446B1 (en) | 2015-08-28 | 2016-12-06 | The General Hospital Corporation | Engineered CRISPR-Cas9 nucleases |
US9926546B2 (en) | 2015-08-28 | 2018-03-27 | The General Hospital Corporation | Engineered CRISPR-Cas9 nucleases |
JP2018530536A (ja) | 2015-09-11 | 2018-10-18 | ザ ジェネラル ホスピタル コーポレイション | ヌクレアーゼDSBの完全照合およびシーケンシング(FIND−seq) |
EP3786294A1 (en) | 2015-09-24 | 2021-03-03 | Editas Medicine, Inc. | Use of exonucleases to improve crispr/cas-mediated genome editing |
US11286480B2 (en) | 2015-09-28 | 2022-03-29 | North Carolina State University | Methods and compositions for sequence specific antimicrobials |
JP2018529353A (ja) | 2015-09-30 | 2018-10-11 | ザ ジェネラル ホスピタル コーポレイション | 配列決定法による切断事象の包括的生体外報告(CIRCLE−seq) |
CN105331627B (zh) * | 2015-09-30 | 2019-04-02 | 华中农业大学 | 一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法 |
US11970710B2 (en) | 2015-10-13 | 2024-04-30 | Duke University | Genome engineering with Type I CRISPR systems in eukaryotic cells |
EP3365357B1 (en) | 2015-10-23 | 2024-02-14 | President and Fellows of Harvard College | Evolved cas9 proteins for gene editing |
ES2699848T3 (es) | 2015-10-23 | 2019-02-13 | Caribou Biosciences Inc | Acido nucleico CRISPR clase 2 de tipo cruzado modificado que se dirige a ácidos nucleicos |
JP6882282B2 (ja) | 2015-11-03 | 2021-06-02 | プレジデント アンド フェローズ オブ ハーバード カレッジ | 三次元核酸含有マトリックスの立体撮像のための方法と装置 |
WO2017112620A1 (en) | 2015-12-22 | 2017-06-29 | North Carolina State University | Methods and compositions for delivery of crispr based antimicrobials |
BR112018013663A2 (pt) | 2016-01-11 | 2019-01-22 | Univ Leland Stanford Junior | proteínas quiméricas e métodos de imunoterapia |
MY196175A (en) | 2016-01-11 | 2023-03-20 | Univ Leland Stanford Junior | Chimeric Proteins And Methods Of Regulating Gene Expression |
CN108884448B (zh) | 2016-01-27 | 2022-12-30 | 昂克诺斯公司 | 溶瘤病毒载体及其用途 |
EP3433364A1 (en) | 2016-03-25 | 2019-01-30 | Editas Medicine, Inc. | Systems and methods for treating alpha 1-antitrypsin (a1at) deficiency |
US11597924B2 (en) | 2016-03-25 | 2023-03-07 | Editas Medicine, Inc. | Genome editing systems comprising repair-modulating enzyme molecules and methods of their use |
WO2017180694A1 (en) | 2016-04-13 | 2017-10-19 | Editas Medicine, Inc. | Cas9 fusion molecules gene editing systems, and methods of use thereof |
EP3449016A4 (en) | 2016-04-25 | 2019-10-02 | President and Fellows of Harvard College | HYBRIDIZATION CHAIN REACTION PROCESS FOR IN-SITU MOLECULE DETECTION |
EP3458595A2 (en) | 2016-05-18 | 2019-03-27 | Amyris, Inc. | Compositions and methods for genomic integration of nucleic acids into exogenous landing pads |
EP3907286A1 (en) | 2016-06-02 | 2021-11-10 | Sigma-Aldrich Co., LLC | Using programmable dna binding proteins to enhance targeted genome modification |
US10767175B2 (en) | 2016-06-08 | 2020-09-08 | Agilent Technologies, Inc. | High specificity genome editing using chemically modified guide RNAs |
EP3472311A4 (en) * | 2016-06-17 | 2020-03-04 | Montana State University | BIDIRECTIONAL TARGETING FOR GENOMEDITATION |
CA3032822A1 (en) | 2016-08-02 | 2018-02-08 | Editas Medicine, Inc. | Compositions and methods for treating cep290 associated disease |
AU2017306676B2 (en) | 2016-08-03 | 2024-02-22 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US11078481B1 (en) | 2016-08-03 | 2021-08-03 | KSQ Therapeutics, Inc. | Methods for screening for cancer targets |
AU2017308889B2 (en) | 2016-08-09 | 2023-11-09 | President And Fellows Of Harvard College | Programmable Cas9-recombinase fusion proteins and uses thereof |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11078483B1 (en) | 2016-09-02 | 2021-08-03 | KSQ Therapeutics, Inc. | Methods for measuring and improving CRISPR reagent function |
US20190225974A1 (en) | 2016-09-23 | 2019-07-25 | BASF Agricultural Solutions Seed US LLC | Targeted genome optimization in plants |
CN110290813A (zh) | 2016-10-14 | 2019-09-27 | 通用医疗公司 | 表观遗传学调控的位点特异性核酸酶 |
SG11201903089RA (en) | 2016-10-14 | 2019-05-30 | Harvard College | Aav delivery of nucleobase editors |
WO2018075664A1 (en) | 2016-10-18 | 2018-04-26 | Regents Of The University Of Minnesota | Tumor infiltrating lymphocytes and methods of therapy |
US9816093B1 (en) | 2016-12-06 | 2017-11-14 | Caribou Biosciences, Inc. | Engineered nucleic acid-targeting nucleic acids |
WO2018119359A1 (en) | 2016-12-23 | 2018-06-28 | President And Fellows Of Harvard College | Editing of ccr5 receptor gene to protect against hiv infection |
US11230710B2 (en) | 2017-01-09 | 2022-01-25 | Aposense Ltd | Compounds and methods for trans-membrane delivery of molecules |
US11866699B2 (en) | 2017-02-10 | 2024-01-09 | University Of Washington | Genome editing reagents and their use |
WO2018165504A1 (en) | 2017-03-09 | 2018-09-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
EP3596217A1 (en) | 2017-03-14 | 2020-01-22 | Editas Medicine, Inc. | Systems and methods for the treatment of hemoglobinopathies |
WO2018170436A1 (en) | 2017-03-16 | 2018-09-20 | Jacobs Farm Del Cabo | Basil with high tolerance to downy mildew |
IL306092A (en) | 2017-03-23 | 2023-11-01 | Harvard College | Nucleic base editors that include nucleic acid programmable DNA binding proteins |
PT3526324T (pt) | 2017-03-28 | 2021-10-20 | Locanabio Inc | Proteína associada a crispr (cas) |
WO2018195545A2 (en) | 2017-04-21 | 2018-10-25 | The General Hospital Corporation | Variants of cpf1 (cas12a) with altered pam specificity |
EP3615672A1 (en) | 2017-04-28 | 2020-03-04 | Editas Medicine, Inc. | Methods and systems for analyzing guide rna molecules |
EP3622070A2 (en) | 2017-05-10 | 2020-03-18 | Editas Medicine, Inc. | Crispr/rna-guided nuclease systems and methods |
WO2018209320A1 (en) | 2017-05-12 | 2018-11-15 | President And Fellows Of Harvard College | Aptazyme-embedded guide rnas for use with crispr-cas9 in genome editing and transcriptional activation |
WO2018218166A1 (en) | 2017-05-25 | 2018-11-29 | The General Hospital Corporation | Using split deaminases to limit unwanted off-target base editor deamination |
EP3635104A1 (en) | 2017-06-09 | 2020-04-15 | Editas Medicine, Inc. | Engineered cas9 nucleases |
WO2018232356A1 (en) | 2017-06-15 | 2018-12-20 | The Regents Of The University Of California | Targeted non-viral dna insertions |
EP3650545A4 (en) | 2017-06-20 | 2021-03-31 | Jiangsu Hengrui Medicine Co., Ltd. | METHOD OF INACTIVATION OF A TARGET GENE IN T CELLS IN VITRO AND ARNCR USED IN THE PROCESS |
WO2019006418A2 (en) | 2017-06-30 | 2019-01-03 | Intima Bioscience, Inc. | ADENO-ASSOCIATED VIRAL VECTORS FOR GENE THERAPY |
WO2019014564A1 (en) | 2017-07-14 | 2019-01-17 | Editas Medicine, Inc. | SYSTEMS AND METHODS OF TARGETED INTEGRATION AND GENOME EDITING AND DETECTION THEREOF WITH INTEGRATED PRIMING SITES |
EP3658165A4 (en) | 2017-07-26 | 2021-09-01 | Oncorus, Inc. | ONCOLYTIC VIRUS VECTORS AND USES THEREOF |
JP2020534795A (ja) | 2017-07-28 | 2020-12-03 | プレジデント アンド フェローズ オブ ハーバード カレッジ | ファージによって支援される連続的進化(pace)を用いて塩基編集因子を進化させるための方法および組成物 |
JP2020532967A (ja) | 2017-08-23 | 2020-11-19 | ザ ジェネラル ホスピタル コーポレイション | 変更されたPAM特異性を有する遺伝子操作されたCRISPR−Cas9ヌクレアーゼ |
WO2019139645A2 (en) | 2017-08-30 | 2019-07-18 | President And Fellows Of Harvard College | High efficiency base editors comprising gam |
US11917978B2 (en) | 2017-09-21 | 2024-03-05 | The Conard Pyle Company | Miniature rose plant named ‘meibenbino’ |
US11252928B2 (en) | 2017-09-21 | 2022-02-22 | The Condard-Pyle Company | Miniature rose plant named ‘Meibenbino’ |
EP3694993A4 (en) | 2017-10-11 | 2021-10-13 | The General Hospital Corporation | METHOD OF DETECTING A SITE-SPECIFIC AND UNDESIRED GENOMIC DESAMINATION INDUCED BY BASE EDITING TECHNOLOGIES |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
WO2019084552A1 (en) | 2017-10-27 | 2019-05-02 | The Regents Of The University Of California | TARGETED REPLACEMENT OF ENDOGENIC T CELL RECEPTORS |
WO2019138083A1 (en) | 2018-01-12 | 2019-07-18 | Basf Se | Gene underlying the number of spikelets per spike qtl in wheat on chromosome 7a |
US20210093679A1 (en) | 2018-02-05 | 2021-04-01 | Novome Biotechnologies, Inc. | Engineered gut microbes and uses thereof |
WO2019186275A1 (en) * | 2018-03-27 | 2019-10-03 | G+Flas Life Sciences | Sequence-specific in vivo cell targeting |
KR102023577B1 (ko) * | 2018-03-27 | 2019-09-24 | (주)지플러스 생명과학 | 가이드 rna 및 엔도뉴클레아제를 유효성분으로 포함하는 암 치료용 약학적 조성물 |
WO2019204378A1 (en) | 2018-04-17 | 2019-10-24 | The General Hospital Corporation | Sensitive in vitro assays for substrate preferences and sites of nucleic acid binding, modifying, and cleaving agents |
GB2589246A (en) | 2018-05-16 | 2021-05-26 | Synthego Corp | Methods and systems for guide RNA design and use |
FR3082208A1 (fr) * | 2018-06-11 | 2019-12-13 | Fondation Mediterranee Infection | Methode de modification d'une sequence cible d'acide nucleique d'une cellule hote |
US20210102183A1 (en) * | 2018-06-13 | 2021-04-08 | Caribou Biosciences, Inc. | Engineered cascade components and cascade complexes |
US10227576B1 (en) | 2018-06-13 | 2019-03-12 | Caribou Biosciences, Inc. | Engineered cascade components and cascade complexes |
WO2019246555A1 (en) * | 2018-06-21 | 2019-12-26 | Cornell University | Type i crispr system as a tool for genome editing |
EP3844273A1 (en) | 2018-08-29 | 2021-07-07 | Amyris, Inc. | Cells and methods for selection based assay |
GB201815820D0 (en) | 2018-09-28 | 2018-11-14 | Univ Wageningen | Off-target activity inhibitors for guided endonucleases |
US20220177943A1 (en) * | 2018-10-01 | 2022-06-09 | Rodolphe Barrangou | Recombinant type i crispr-cas system and uses thereof for screening for variant cells |
US10711267B2 (en) | 2018-10-01 | 2020-07-14 | North Carolina State University | Recombinant type I CRISPR-Cas system |
WO2020092725A1 (en) * | 2018-11-01 | 2020-05-07 | Montana State University | Gene modulation with crispr system type i |
US20210388333A1 (en) * | 2018-11-09 | 2021-12-16 | Inari Agriculture, Inc. | Rna-guided nucleases and dna binding proteins |
JP2022514493A (ja) | 2018-12-14 | 2022-02-14 | パイオニア ハイ-ブレッド インターナショナル, インコーポレイテッド | ゲノム編集のための新規なcrispr-casシステム |
US11946040B2 (en) | 2019-02-04 | 2024-04-02 | The General Hospital Corporation | Adenine DNA base editor variants with reduced off-target RNA editing |
WO2020176389A1 (en) | 2019-02-25 | 2020-09-03 | Caribou Biosciences, Inc. | Plasmids for gene editing |
JP2022526908A (ja) | 2019-03-19 | 2022-05-27 | ザ ブロード インスティテュート,インコーポレーテッド | 編集ヌクレオチド配列を編集するための方法および組成物 |
US20220186263A1 (en) | 2019-04-05 | 2022-06-16 | Osaka University | Method for producing knock-in cell |
CN110438142A (zh) * | 2019-05-13 | 2019-11-12 | 安徽大学 | 一种基于I-B-Svi型CRISPR-Cas系统中SviCas5-6-7的转录调控方法 |
WO2021092210A1 (en) * | 2019-11-06 | 2021-05-14 | Locus Biosciences, Inc. | Phage compositions comprising crispr-cas systems and methods of use thereof |
US20230022635A1 (en) * | 2019-12-31 | 2023-01-26 | Inari Agriculture Technology, Inc. | Delivery of biological molecules to plant cells |
AU2021267940A1 (en) | 2020-05-08 | 2022-12-08 | President And Fellows Of Harvard College | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
CN111849978B (zh) * | 2020-06-11 | 2022-04-15 | 中山大学 | 一种基于Type I-F CRISPR/Cas的染色质成像方法和染色质成像系统 |
CN111979240B (zh) * | 2020-06-11 | 2022-04-15 | 中山大学 | 一种基于Type I-F CRISPR/Cas的基因表达调控方法和调控系统 |
IL301396A (en) | 2020-09-30 | 2023-05-01 | Nobell Foods Inc | Recombinant milk proteins and food compositions containing them |
US10894812B1 (en) | 2020-09-30 | 2021-01-19 | Alpine Roads, Inc. | Recombinant milk proteins |
US11944063B2 (en) | 2020-09-30 | 2024-04-02 | Spring Meadow Nursery, Inc. | Hydrangea ‘SMNHPH’ |
US10947552B1 (en) | 2020-09-30 | 2021-03-16 | Alpine Roads, Inc. | Recombinant fusion proteins for producing milk proteins in plants |
EP3984355B1 (en) | 2020-10-16 | 2024-05-15 | Klemm & Sohn GmbH & Co. KG | Double-flowering dwarf calibrachoa |
US11155884B1 (en) | 2020-10-16 | 2021-10-26 | Klemm & Sohn Gmbh & Co. Kg | Double-flowering dwarf Calibrachoa |
WO2022093977A1 (en) | 2020-10-30 | 2022-05-05 | Fortiphyte, Inc. | Pathogen resistance in plants |
US20230407350A1 (en) | 2020-11-10 | 2023-12-21 | Industrial Microbes, Inc. | Microorganisms capable of producing poly(hiba) from feedstock |
CA3222023A1 (en) | 2021-06-01 | 2022-12-08 | Arbor Biotechnologies, Inc. | Gene editing systems comprising a crispr nuclease and uses thereof |
WO2023039491A2 (en) * | 2021-09-09 | 2023-03-16 | Proof Diagnostics, Inc. | Coronavirus rapid diagnostics |
AU2022343300A1 (en) | 2021-09-10 | 2024-04-18 | Agilent Technologies, Inc. | Guide rnas with chemical modification for prime editing |
US20230279442A1 (en) | 2021-12-15 | 2023-09-07 | Versitech Limited | Engineered cas9-nucleases and method of use thereof |
CN117384867A (zh) * | 2022-09-16 | 2024-01-12 | 北京普译生物科技有限公司 | 一种经修饰的Cas3移位酶及其应用 |
CN115851664B (zh) * | 2022-09-19 | 2023-08-25 | 中国药科大学 | 一种I-B型CRISPR-Cascade-Cas3基因编辑系统及应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100076057A1 (en) * | 2008-09-23 | 2010-03-25 | Northwestern University | TARGET DNA INTERFERENCE WITH crRNA |
WO2010054108A2 (en) * | 2008-11-06 | 2010-05-14 | University Of Georgia Research Foundation, Inc. | Cas6 polypeptides and methods of use |
EP2341149A1 (en) * | 2005-08-26 | 2011-07-06 | Danisco A/S | Use of CRISPR associated genes (CAS) |
WO2011097036A1 (en) * | 2010-02-08 | 2011-08-11 | Sangamo Biosciences, Inc. | Engineered cleavage half-domains |
Family Cites Families (120)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5034506A (en) | 1985-03-15 | 1991-07-23 | Anti-Gene Development Group | Uncharged morpholino-based polymers having achiral intersubunit linkages |
WO1988008450A1 (en) | 1987-05-01 | 1988-11-03 | Birdwell Finlayson | Gene therapy for metabolite disorders |
US5350689A (en) | 1987-05-20 | 1994-09-27 | Ciba-Geigy Corporation | Zea mays plants and transgenic Zea mays plants regenerated from protoplasts or protoplast-derived cells |
US5767367A (en) | 1990-06-23 | 1998-06-16 | Hoechst Aktiengesellschaft | Zea mays (L.) with capability of long term, highly efficient plant regeneration including fertile transgenic maize plants having a heterologous gene, and their preparation |
US5489677A (en) | 1990-07-27 | 1996-02-06 | Isis Pharmaceuticals, Inc. | Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms |
US5602240A (en) | 1990-07-27 | 1997-02-11 | Ciba Geigy Ag. | Backbone modified oligonucleotide analogs |
FR2688514A1 (fr) | 1992-03-16 | 1993-09-17 | Centre Nat Rech Scient | Adenovirus recombinants defectifs exprimant des cytokines et medicaments antitumoraux les contenant. |
US5625048A (en) | 1994-11-10 | 1997-04-29 | The Regents Of The University Of California | Modified green fluorescent proteins |
US5968738A (en) | 1995-12-06 | 1999-10-19 | The Board Of Trustees Of The Leland Stanford Junior University | Two-reporter FACS analysis of mammalian cells using green fluorescent proteins |
US20020182673A1 (en) | 1998-05-15 | 2002-12-05 | Genentech, Inc. | IL-17 homologous polypedies and therapeutic uses thereof |
US6306610B1 (en) | 1998-09-18 | 2001-10-23 | Massachusetts Institute Of Technology | Biological applications of quantum dots |
EP1147209A2 (en) | 1999-02-03 | 2001-10-24 | The Children's Medical Center Corporation | Gene repair involving the induction of double-stranded dna cleavage at a chromosomal target site |
AU784151B2 (en) | 1999-12-28 | 2006-02-09 | Ribonomics, Inc. | Methods for isolating and characterizing endogenous mRNA-protein (mRNP) complexes |
US20020119570A1 (en) | 2000-09-25 | 2002-08-29 | Kyonggeun Yoon | Targeted gene correction by single-stranded oligodeoxynucleotides |
MXPA03003690A (es) | 2000-10-27 | 2004-05-05 | Chiron Spa | Acidos nucleicos y proteinas de los grupos a y b de estreptococos. |
US20060253913A1 (en) | 2001-12-21 | 2006-11-09 | Yue-Jin Huang | Production of hSA-linked butyrylcholinesterases in transgenic mammals |
CA2474486C (en) | 2002-01-23 | 2013-05-14 | The University Of Utah Research Foundation | Targeted chromosomal mutagenesis using zinc finger nucleases |
US20030232410A1 (en) | 2002-03-21 | 2003-12-18 | Monika Liljedahl | Methods and compositions for using zinc finger endonucleases to enhance homologous recombination |
US7539579B2 (en) | 2002-04-09 | 2009-05-26 | Beattie Kenneth L | Oligonucleotide probes for genosensor chips |
EP2806025B1 (en) | 2002-09-05 | 2019-04-03 | California Institute of Technology | Use of zinc finger nucleases to stimulate gene targeting |
US20120196370A1 (en) | 2010-12-03 | 2012-08-02 | Fyodor Urnov | Methods and compositions for targeted genomic deletion |
US7972854B2 (en) | 2004-02-05 | 2011-07-05 | Sangamo Biosciences, Inc. | Methods and compositions for targeted cleavage and recombination |
CN101426931A (zh) * | 2004-04-07 | 2009-05-06 | 安克塞斯生物公司 | 核酸检测系统 |
US7919277B2 (en) | 2004-04-28 | 2011-04-05 | Danisco A/S | Detection and typing of bacterial strains |
US7892224B2 (en) | 2005-06-01 | 2011-02-22 | Brainlab Ag | Inverse catheter planning |
KR101419729B1 (ko) | 2005-07-26 | 2014-07-17 | 상가모 바이오사이언스 인코포레이티드 | 외래 핵산 서열의 표적화된 통합 및 발현 |
ATE513053T1 (de) | 2006-05-10 | 2011-07-15 | Deinove Sa | Verfahren zur chromosomenmanipulation unter verwendung eines neuen dna-reparatursystems |
ATE530669T1 (de) | 2006-05-19 | 2011-11-15 | Danisco | Markierte mikroorganismen und entsprechende markierungsverfahren |
WO2007139982A2 (en) | 2006-05-25 | 2007-12-06 | Sangamo Biosciences, Inc. | Methods and compositions for gene inactivation |
PL2034848T3 (pl) | 2006-06-16 | 2017-08-31 | Dupont Nutrition Bioscences Aps | Bakteria Streptococcus thermophilus |
WO2008019123A2 (en) | 2006-08-04 | 2008-02-14 | Georgia State University Research Foundation, Inc. | Enzyme sensors, methods for preparing and using such sensors, and methods of detecting protease activity |
WO2008108989A2 (en) | 2007-03-02 | 2008-09-12 | Danisco A/S | Cultures with improved phage resistance |
EP2142673B1 (en) * | 2007-04-05 | 2013-05-22 | Johnson & Johnson Research Pty Limited | Nucleic acid enzymes and complexes and methods for their use |
FR2925918A1 (fr) | 2007-12-28 | 2009-07-03 | Pasteur Institut | Typage et sous-typage moleculaire de salmonella par identification des sequences nucleotidiques variables des loci crispr |
US8546553B2 (en) | 2008-07-25 | 2013-10-01 | University Of Georgia Research Foundation, Inc. | Prokaryotic RNAi-like system and methods of use |
CN102159722B (zh) | 2008-08-22 | 2014-09-03 | 桑格摩生物科学股份有限公司 | 用于靶向单链切割和靶向整合的方法和组合物 |
EP3208339B1 (en) | 2008-09-15 | 2019-05-01 | The Children's Medical Center Corporation | Modulation of bcl11a for treatment of hemoglobinopathies |
MX337838B (es) | 2008-11-07 | 2016-03-22 | Dupont Nutrition Biosci Aps | Secuencias de repetidos palindromicos cortos regularmente intercalados agrupados de bifidobacterias. |
PT2367938E (pt) | 2008-12-12 | 2014-09-05 | Dupont Nutrition Biosci Aps | Agrupamento genético de estirpes de streptococcus thermophilus com propriedades reológicas únicas para a fermentação de lacticínios |
WO2010075424A2 (en) | 2008-12-22 | 2010-07-01 | The Regents Of University Of California | Compositions and methods for downregulating prokaryotic genes |
CA2756833C (en) | 2009-04-09 | 2019-11-19 | Sangamo Biosciences, Inc. | Targeted integration into stem cells |
ES2786039T3 (es) | 2009-04-30 | 2020-10-08 | Ospedale San Raffaele Srl | Vector génico |
JP2012529287A (ja) | 2009-06-11 | 2012-11-22 | トゥールゲン インコーポレイション | 部位−特異的ヌクレアーゼを用いた標的ゲノムの再配列 |
SG177711A1 (en) | 2009-07-24 | 2012-02-28 | Sigma Aldrich Co Llc | Method for genome editing |
US20120192298A1 (en) | 2009-07-24 | 2012-07-26 | Sigma Aldrich Co. Llc | Method for genome editing |
US9234016B2 (en) | 2009-07-28 | 2016-01-12 | Sangamo Biosciences, Inc. | Engineered zinc finger proteins for treating trinucleotide repeat disorders |
WO2011019385A1 (en) | 2009-08-11 | 2011-02-17 | Sangamo Biosciences, Inc. | Organisms homozygous for targeted modification |
EP2292731B1 (en) | 2009-08-13 | 2018-04-04 | DuPont Nutrition Biosciences ApS | Method for preparing complex cultures |
EA201290168A1 (ru) | 2009-09-25 | 2013-10-30 | Басф Плант Сайенс Компани Гмбх | Растения, обладающие повышенными урожайностными свойствами, и способ создания таких растений |
US20110105364A1 (en) | 2009-11-02 | 2011-05-05 | Nugen Technologies, Inc. | Compositions and methods for targeted nucleic acid sequence selection and amplification |
BR112012012444A2 (pt) | 2009-11-27 | 2015-09-22 | Basf Plant Science Co Gmbh | "endonuclease quimérica, polinucleotídeo isolado, cassete de expressão, vetor, organismo não humano, método para fornecer uma endonuclease quimérica, método para recombinação homóloga de polinucleotídeos, método para mutação alvejada de polinucleotídeosa e método para recombinação homóloga ou mutação alvejada" |
US20110294114A1 (en) | 2009-12-04 | 2011-12-01 | Cincinnati Children's Hospital Medical Center | Optimization of determinants for successful genetic correction of diseases, mediated by hematopoietic stem cells |
SG181601A1 (en) | 2009-12-10 | 2012-07-30 | Univ Minnesota | Tal effector-mediated dna modification |
US20110203012A1 (en) | 2010-01-21 | 2011-08-18 | Dotson Stanton B | Methods and compositions for use of directed recombination in plant breeding |
US9255259B2 (en) | 2010-02-09 | 2016-02-09 | Sangamo Biosciences, Inc. | Targeted genomic modification with partially single-stranded donor molecules |
US10087431B2 (en) | 2010-03-10 | 2018-10-02 | The Regents Of The University Of California | Methods of generating nucleic acid fragments |
US20130059388A1 (en) | 2010-04-13 | 2013-03-07 | Sigma-Aldrich Co., Llc | Methods for generating endogenously tagged proteins |
SG185481A1 (en) | 2010-05-10 | 2012-12-28 | Univ California | Endoribonuclease compositions and methods of use thereof |
EP2571512B1 (en) | 2010-05-17 | 2017-08-23 | Sangamo BioSciences, Inc. | Novel dna-binding proteins and uses thereof |
WO2011156430A2 (en) | 2010-06-07 | 2011-12-15 | Fred Hutchinson Cancer Research Center | Generation and expression of engineered i-onui endonuclease and its homologues and uses thereof |
EP2392208B1 (en) * | 2010-06-07 | 2016-05-04 | Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Fusion proteins comprising a DNA-binding domain of a Tal effector protein and a non-specific cleavage domain of a restriction nuclease and their use |
JP2013537410A (ja) | 2010-07-23 | 2013-10-03 | シグマ−アルドリッチ・カンパニー・リミテッド・ライアビリティ・カンパニー | 標的化エンドヌクレアーゼおよび一本鎖核酸を用いたゲノム編集 |
EP2601611B1 (en) | 2010-08-02 | 2020-12-09 | Integrated Dna Technologies, Inc. | Methods for predicting stability and melting temperatures of nucleic acid duplexes |
EP2630156B1 (en) | 2010-10-20 | 2018-08-22 | DuPont Nutrition Biosciences ApS | Lactococcus crispr-cas sequences |
KR20120096395A (ko) | 2011-02-22 | 2012-08-30 | 주식회사 툴젠 | 뉴클레아제에 의해 유전자 변형된 세포를 농축시키는 방법 |
US20140123330A1 (en) | 2012-10-30 | 2014-05-01 | Recombinetics, Inc. | Control of sexual maturation in animals |
CA2834375C (en) | 2011-04-27 | 2020-07-14 | Amyris, Inc. | Methods for genomic modification |
WO2012164565A1 (en) | 2011-06-01 | 2012-12-06 | Yeda Research And Development Co. Ltd. | Compositions and methods for downregulating prokaryotic genes |
US8927218B2 (en) | 2011-06-27 | 2015-01-06 | Flir Systems, Inc. | Methods and compositions for segregating target nucleic acid from mixed nucleic acid samples |
CN103917644A (zh) | 2011-09-21 | 2014-07-09 | 桑格摩生物科学股份有限公司 | 调控转基因表达的方法和组合物 |
US8450107B1 (en) | 2011-11-30 | 2013-05-28 | The Broad Institute Inc. | Nucleotide-specific recognition sequences for designer TAL effectors |
IN2014MN00974A (ja) | 2011-12-16 | 2015-04-24 | Targetgene Biotechnologies Ltd | |
GB201122458D0 (en) | 2011-12-30 | 2012-02-08 | Univ Wageningen | Modified cascade ribonucleoproteins and uses thereof |
EP3272356A1 (en) | 2012-02-24 | 2018-01-24 | Fred Hutchinson Cancer Research Center | Compositions and methods for the treatment of hemoglobinopathies |
JP6490426B2 (ja) | 2012-02-29 | 2019-03-27 | サンガモ セラピューティクス, インコーポレイテッド | ハンチントン病を治療するための方法および組成物 |
WO2013141680A1 (en) | 2012-03-20 | 2013-09-26 | Vilnius University | RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX |
US9637739B2 (en) | 2012-03-20 | 2017-05-02 | Vilnius University | RNA-directed DNA cleavage by the Cas9-crRNA complex |
AU2013204327B2 (en) | 2012-04-20 | 2016-09-01 | Aviagen | Cell transfection method |
US11518997B2 (en) | 2012-04-23 | 2022-12-06 | BASF Agricultural Solutions Seed US LLC | Targeted genome engineering in plants |
MX369788B (es) | 2012-05-02 | 2019-11-21 | Dow Agrosciences Llc | Modificacion dirigida de deshidrogenasa de malato. |
RU2650819C2 (ru) | 2012-05-07 | 2018-04-17 | Сангамо Терапьютикс, Инк. | Способы и композиции для опосредованной нуклеазой направленной интеграции трансгенов |
US11120889B2 (en) | 2012-05-09 | 2021-09-14 | Georgia Tech Research Corporation | Method for synthesizing a nuclease with reduced off-site cleavage |
LT3401400T (lt) * | 2012-05-25 | 2019-06-10 | The Regents Of The University Of California | Būdai ir kompozicijos, skirtos rnr molekulės nukreipiamai tikslinės dnr modifikacijai ir rnr molekulės nukreipiamam transkripcijos moduliavimui |
MX2014014650A (es) | 2012-05-30 | 2015-10-14 | Univ Washington | Minivectores superenrollados como una herramienta para la reparacion, alteración y reemplazo de ácido desoxirribonucleico. |
WO2013188037A2 (en) | 2012-06-11 | 2013-12-19 | Agilent Technologies, Inc | Method of adaptor-dimer subtraction using a crispr cas6 protein |
CN104540382A (zh) | 2012-06-12 | 2015-04-22 | 弗·哈夫曼-拉罗切有限公司 | 用于产生条件性敲除等位基因的方法和组合物 |
EP2674501A1 (en) | 2012-06-14 | 2013-12-18 | Agence nationale de sécurité sanitaire de l'alimentation,de l'environnement et du travail | Method for detecting and identifying enterohemorrhagic Escherichia coli |
US9688971B2 (en) | 2012-06-15 | 2017-06-27 | The Regents Of The University Of California | Endoribonuclease and methods of use thereof |
EP2861737B1 (en) | 2012-06-19 | 2019-04-17 | Regents Of The University Of Minnesota | Gene targeting in plants using dna viruses |
HUE041553T2 (hu) | 2012-07-11 | 2019-05-28 | Sangamo Therapeutics Inc | Lizoszomális tárolási betegségek (LSD) kezelése és génterápia |
CA2878037C (en) | 2012-07-11 | 2021-08-31 | Sangamo Biosciences, Inc. | Methods and compositions for delivery of biologics |
PT3494997T (pt) | 2012-07-25 | 2019-12-05 | Massachusetts Inst Technology | Proteínas de ligação a adn indutíveis e ferramentas de perturbação do genoma e aplicações destas |
ES2812599T3 (es) | 2012-08-29 | 2021-03-17 | Sangamo Therapeutics Inc | Procedimientos y composiciones para el tratamiento de una afección genética |
UA119135C2 (uk) | 2012-09-07 | 2019-05-10 | ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі | Спосіб отримання трансгенної рослини |
CN105264067B (zh) | 2012-09-07 | 2020-11-10 | 美国陶氏益农公司 | Fad3性能基因座及相应的能够诱导靶向断裂的靶位点特异性结合蛋白 |
UA118090C2 (uk) | 2012-09-07 | 2018-11-26 | ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі | Спосіб інтегрування послідовності нуклеїнової кислоти, що представляє інтерес, у ген fad2 у клітині сої та специфічний для локусу fad2 білок, що зв'язується, здатний індукувати спрямований розрив |
EP2906602B1 (en) | 2012-10-12 | 2019-01-16 | The General Hospital Corporation | Transcription activator-like effector (tale) - lysine-specific demethylase 1 (lsd1) fusion proteins |
DE202013012597U1 (de) | 2012-10-23 | 2017-11-21 | Toolgen, Inc. | Zusammensetzung zum Spalten einer Ziel-DNA, umfassend eine für die Ziel-DNA spezifische guide-RNA und eine Cas-Protein-codierende Nukleinsäure oder ein Cas-Protein, sowie deren Verwendung |
US20150291967A1 (en) | 2012-10-31 | 2015-10-15 | Luc Mathis | Coupling herbicide resistance with targeted insertion of transgenes in plants |
US20140127752A1 (en) | 2012-11-07 | 2014-05-08 | Zhaohui Zhou | Method, composition, and reagent kit for targeted genomic enrichment |
EP3138911B1 (en) * | 2012-12-06 | 2018-12-05 | Sigma Aldrich Co. LLC | Crispr-based genome modification and regulation |
ES2658401T3 (es) | 2012-12-12 | 2018-03-09 | The Broad Institute, Inc. | Suministro, modificación y optimización de sistemas, métodos y composiciones para la manipulación de secuencias y aplicaciones terapéuticas |
EP2931892B1 (en) | 2012-12-12 | 2018-09-12 | The Broad Institute, Inc. | Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof |
EP2931899A1 (en) | 2012-12-12 | 2015-10-21 | The Broad Institute, Inc. | Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof |
WO2014093694A1 (en) | 2012-12-12 | 2014-06-19 | The Broad Institute, Inc. | Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes |
ES2576128T3 (es) | 2012-12-12 | 2016-07-05 | The Broad Institute, Inc. | Modificación por tecnología genética y optimización de sistemas, métodos y composiciones para la manipulación de secuencias con dominios funcionales |
US8697359B1 (en) | 2012-12-12 | 2014-04-15 | The Broad Institute, Inc. | CRISPR-Cas systems and methods for altering expression of gene products |
PL2784162T3 (pl) | 2012-12-12 | 2016-01-29 | Broad Inst Inc | Opracowanie systemów, metod oraz zoptymalizowanych kompozycji przewodnikowych do manipulacji sekwencyjnej |
EP4234696A3 (en) | 2012-12-12 | 2023-09-06 | The Broad Institute Inc. | Crispr-cas component systems, methods and compositions for sequence manipulation |
PT2921557T (pt) | 2012-12-12 | 2016-10-19 | Massachusetts Inst Technology | Engenharia de sistemas, métodos e composições guia otimizadas para a manipulação de sequências |
DK2898075T3 (en) | 2012-12-12 | 2016-06-27 | Broad Inst Inc | CONSTRUCTION AND OPTIMIZATION OF IMPROVED SYSTEMS, PROCEDURES AND ENZYME COMPOSITIONS FOR SEQUENCE MANIPULATION |
CA3081054A1 (en) | 2012-12-17 | 2014-06-26 | President And Fellows Of Harvard College | Rna-guided human genome engineering |
WO2014104878A1 (en) | 2012-12-27 | 2014-07-03 | Keygene N.V. | Method for removing genetic linkage in a plant |
AU2014207618A1 (en) | 2013-01-16 | 2015-08-06 | Emory University | Cas9-nucleic acid complexes and uses related thereto |
CN103233028B (zh) | 2013-01-25 | 2015-05-13 | 南京徇齐生物技术有限公司 | 一种无物种限制无生物安全性问题的真核生物基因打靶方法及螺旋结构dna序列 |
CA2906553C (en) * | 2013-03-15 | 2022-08-02 | The General Hospital Corporation | Rna-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci |
CN103224947B (zh) | 2013-04-28 | 2015-06-10 | 陕西师范大学 | 一种基因打靶系统 |
EP3417880A1 (en) * | 2013-06-05 | 2018-12-26 | Duke University | Rna-guided gene editing and gene regulation |
CN103343120B (zh) | 2013-07-04 | 2015-03-04 | 中国科学院遗传与发育生物学研究所 | 一种小麦基因组定点改造方法 |
US10227576B1 (en) * | 2018-06-13 | 2019-03-12 | Caribou Biosciences, Inc. | Engineered cascade components and cascade complexes |
-
2011
- 2011-12-30 GB GB201122458A patent/GB201122458D0/en not_active Ceased
-
2012
- 2012-12-21 CN CN201280071058.4A patent/CN104321429A/zh active Pending
- 2012-12-21 LT LTEP16169431.0T patent/LT3091072T/lt unknown
- 2012-12-21 CN CN201610081216.4A patent/CN105732816B/zh active Active
- 2012-12-21 JP JP2014549450A patent/JP6408914B2/ja active Active
- 2012-12-21 GB GB1411878.0A patent/GB2512246B/en active Active
- 2012-12-21 EP EP16169431.0A patent/EP3091072B1/en active Active
- 2012-12-21 CA CA2862018A patent/CA2862018C/en active Active
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2341149A1 (en) * | 2005-08-26 | 2011-07-06 | Danisco A/S | Use of CRISPR associated genes (CAS) |
US20100076057A1 (en) * | 2008-09-23 | 2010-03-25 | Northwestern University | TARGET DNA INTERFERENCE WITH crRNA |
WO2010054108A2 (en) * | 2008-11-06 | 2010-05-14 | University Of Georgia Research Foundation, Inc. | Cas6 polypeptides and methods of use |
WO2011097036A1 (en) * | 2010-02-08 | 2011-08-11 | Sangamo Biosciences, Inc. | Engineered cleavage half-domains |
Non-Patent Citations (11)
Title |
---|
BIOLOGY DIRECT (2011) VOL.6, NO.38, PP.1-27, JPN5015002271, ISSN: 0003836032 * |
CARYN R. HALE, MOLECULAR CELL, vol. V45 N3, JPN5015002275, 1 February 2012 (2012-02-01), pages 292 - 302, ISSN: 0003836040 * |
DATABASE DDBJ/EMBL/GENBANK [ONLINE], ACCESSIN NO. ZP_07272643.1 (REFSEQ: ACCESSION NZ_GG657742.1), <, JPN7017003014, ISSN: 0003836031 * |
EDZE R. WESTRA, RNA BIOLOGY, vol. V9 N9, JPN5015002276, 1 September 2012 (2012-09-01), pages 1134 - 1138, ISSN: 0003836041 * |
GENES & DEVELOPMENT (2008) VOL.22, PP.3489-3496, JPN7016002929, ISSN: 0003836036 * |
MAKAROVA KIRA S: "EVOLUTION AND CLASSIFICATION OF THE CRISPR-CAS SYSTEMS", NATURE REVIEWS MICROBIOLOGY, vol. V9 N6, JPN5015002272, 1 June 2011 (2011-06-01), GB, pages 467 - 477, ISSN: 0003836038 * |
NATHANAEL G. LINTNER, JOURNAL OF BIOLOGICAL CHEMISTRY, vol. V286 N24, JPN5015002270, 17 June 2011 (2011-06-17), pages 21643 - 21656, ISSN: 0003836037 * |
NATURE (2011) VOL.477, PP.486-489, JPN5015002269, ISSN: 0003836033 * |
NATURE REVIEWS MICROBIOLOGY (2008) VOL.6, PP.181-186, JPN5015002274, ISSN: 0003836035 * |
NATURE STRUCTURAL & MOLECULAR BIOLOGY (2011) VOL.18, NO.5, PP.529-536, JPN7017003015, ISSN: 0003836034 * |
RITSDELIZ PEREZ-RODRIGUEZ, MOLECULAR MICROBIOLOGY, vol. V79 N3, JPN5015002273, 1 February 2011 (2011-02-01), pages 584 - 599, ISSN: 0003836039 * |
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JP7422128B2 (ja) | 2018-04-03 | 2024-01-25 | ジーフラス ライフ サイエンシズ,インク. | 配列特異的なインビボ細胞標的化 |
JP2021520844A (ja) * | 2018-06-13 | 2021-08-26 | カリブー・バイオサイエンシーズ・インコーポレイテッド | 操作されたカスケード構成要素およびカスケード複合体 |
WO2021149829A1 (ja) * | 2020-01-24 | 2021-07-29 | C4U株式会社 | 試料中の特定のdnaを検出する方法 |
JP6940086B1 (ja) * | 2020-01-24 | 2021-09-22 | C4U株式会社 | 試料中の特定のdnaを検出する方法 |
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