JP2016507244A - Cas9ヌクレアーゼによる卵母細胞における遺伝子編集 - Google Patents
Cas9ヌクレアーゼによる卵母細胞における遺伝子編集 Download PDFInfo
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Abstract
Description
Cas9 mRNA、tracrRNA/crRNAまたはキメラRNA(chRNA)および任意に、合成オリゴデオキシヌクレオチド(ODN)の、ドナー雌から単離した一細胞胚の父型前核へのマイクロインジェクションによるCrispr/Cas9を介した胚に基づく遺伝子ターゲッティングのワークフロー。翻訳時に、Cas9ヌクレアーゼタンパク質は前核に輸入され、標的特異的crRNAおよび一般的tracrRNAと一緒に、または標的特異的chRNAと一緒に、父型および母型ゲノムの標的遺伝子中に二本鎖切断を生じる(図1)。父型ゲノムにおいて、DSBは、いずれか一方が相同組み換えにより突然変異誘発ODNで閉じられる(seal)か、または両方のゲノムにおいて間違いが起こりやすいNHEJ修復により処理され、ノックインまたはノックアウト対立遺伝子を生じる(図2)。マイクロインジェクションされた胚を親雌に移して(upon)、生まれる子孫をPCRおよび配列分析により遺伝子型決定し、生殖系にターゲッティングされた変異またはノックアウト変異を有する創始動物を同定する。かかる創始マウスと野生型マウスの交配により、ヘテロ接合体変異体を生じ、これを交雑してホモ接合体変異体を得る。
プラスミド構築
短いRNAのインビトロ転写のためのDNA構築物pT7-tracrRNA、pT7-crRNA-Rab38、pT7crRNA-Fus、pT7chRNA-Rab38およびpT7-chRNA-Fus(図3)を、DNA合成(Genscript, Piscataway, USA)により得て、プラスミドpUC57にクローニングした。crRNAは、化膿連鎖球菌SF370 II型CRISPR座PAM配列「NGG」の上流に位置するゲノム配列中の標的配列を認識するように設計した。プラスミドpCAG-Cas9-bpA (配列番号:3)は、合成Cas9コーディング領域を含むPacI-MluI断片の連結(Genscript, Piscataway, USA)により、プラスミドpCAG-venus-MluIの対応する部位に構築した。プラスミドpCAG-Cas9-bpAは、N末端およびC末端核局在化配列(NLS)およびFLAGタグ、次いで直線化のためのMluI制限部位の付加により改変された、化膿連鎖球菌II型CRISPR座由来のCas9のコドン最適化コーディング領域の上流にT7 RNAポリメラーゼプロモーターを含む。プラスミドpT7-tracrRNA (配列番号:4)は、指定される配列の上流にT7プロモーターを含み、89ヌクレオチドのtracrRNAのインビトロ転写を可能にする。プラスミドpT7-crRNA-Rab38 (配列番号:5)は指定される102ヌクレオチド配列の上流にT7プロモーターを含み、化膿連鎖球菌II型CRISPR座由来の2つの36nt直接リピート(DR)配列に挟まれたマウスRab38遺伝子のエクソン1由来の30nt標的配列を含むcrRNA-Rab38のインビトロ転写を可能にする。プラスミドpT7-crRNA-Fus (配列番号:8)は、指定される102ヌクレオチド配列の上流にT7プロモーターを含み、化膿連鎖球菌II型CRISPR座由来の2つの36nt DR配列で挟まれたマウスFus遺伝子のエクソン15由来の30nt標的配列を含むcrRNA-Fusのインビトロ転写を可能にする。プラスミドpT7-chRNA-Rab38 (配列番号:6)は、指定される103ヌクレオチド配列の上流にT7プロモーターを含み、マウスRab38遺伝子のエクソン1由来の20nt標的配列ならびにcrRNAおよびtracr RNA由来のキメラRNA配列を含むchRNA-Rab38(図3)のインビトロ転写を可能にする。プラスミドpT7-chRNA-Fus (配列番号:9)は、指定される103ヌクレオチド配列の上流にT7プロモーターを含み、マウスFus遺伝子のエクソン15由来の20nt標的配列ならびにcrRNAおよびtracr RNA由来のキメラRNA配列を含むchRNA-Fus(図3)のインビトロ転写を可能にする。
Cas9 mRNAのインジェクション、tracrRNA/crRNAまたはchRNAおよびターゲッティングODNのインジェクションは、ZFNについて以前に記載されたように実施される(Meyer et al. (2010) Proc Natl Acad Sci U S A 107:15022-6; Meyer et al. (2012) Proc Natl Acad Sci USA 109:9354-9359)。簡潔に、Cas9 mRNA(ポリアデニル化を含む)、tracrRNA/crRNAまたはchRNAs(ポリアデニル化を含まない)は、mMessage mMachine T7 UltraキットおよびMEGAclearキット(Life Technologies, Carlsbad, USA)を使用して、MluI (Cas9)またはAlwI (RNA)を用いて転写された領域の末端で直線化されたプラスミドDNAからのインビトロ転写により調製する。次いでRNAのそれぞれを注入バッファ(10mM Tris、0.1mM EDTA、pH7.2)中に20ng/μlの作業濃度まで希釈した。ターゲッティングオリゴデオキシヌクレオチド(Metabion, Martinsried, Germany)は注入バッファ内に入れ、30ng/μlの作業濃度まで希釈した。適切なRNAをそれぞれの突然変異誘発オリゴデオキシヌクレオチドと混合し、-80℃で保存した。C57BL/6N雄と***過剰(super-ovulated)FVB雌(Charles River, Sulzbach, Germany)の交配により一細胞胚を得た。過剰***3週齢FVB雌は、交配の2日前に2.5IUの妊馬血清(PMS)で処理し、交配日に2.5IUヒト絨毛性ゴナドトロピン(hCG)で処理する。受精した卵母細胞をプラグ陽性(plug positive)雌の卵管から単離し、標準的な手法に従って、M2培地(Sigma-Aldrich Inc Cat. No. M7167)中でマイクロインジェクションした(Nagy A, Gertsenstein M, Vintersten K, Behringer R., 2003. Manipulating the Mouse Embryo. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press)。記載されるように、2段階手法で、胚にターゲッティングODNとRNAの混合物を注入した(Meyer et al. (2010) Proc Natl Acad Sci U S A 107:15022-6; Meyer et al. (2012) Proc Natl Acad Sci USA 109:9354-9359)。簡潔に、トランスジェニックマウスの作製に使用されるように、DNA/RNA混合物の第1のアリコートを、可能な場合はいつでも、大きい方の(雄)前核に注入し、DNAベクターを送達した。前核から注入針を引き抜く際に、DNA/RNA混合物の第2のアリコートを細胞質に注入し、Cas9 mRNAを翻訳機構に直接送達した。Leicaマイクロマニピュレーターおよび顕微鏡およびEppendorf FemtoJet注入デバイスを使用して注入を行った。注入した接合体を、偽妊娠CD1雌マウスに移し、生きた成体マウスを得た。
Wizard Genomic DNA Purification Kit (Promega)のプロトコルに従い、マイクロインジェクション由来のマウスの尾の先端からゲノムDNAを単離した。得られたDNAペレットを100μl 10mM Tris-Cl、pH8.5に懸濁して、室温で一晩インキュベートし、さらなる分析のために4℃で保存した。Rab38遺伝子中の変異についての創始物(founder)の分析のために、PCRプライマーペアRab-for (配列番号:10) (5'-GGCCTCCAGGATGCAGACACC-3')およびRab-rev (配列番号:11) (5'-CCAGCAATGTCCCAGAGCTGC-3')を使用してエクソン1を増幅した。増幅は、95℃ 20秒、60℃ 15秒、72℃ 15秒の30サイクルの25μl反応において、Herculase IIポリメラーゼ(Agilent Technologies)を使用して行った。その後、PCR産物を、10UのSexAIで直接消化し、アガロースゲル上で分析した。陽性と同定された創始物由来の消化されなかった産物を、Qiaquick PCR精製キット(Qiagen)で精製し、pSC-B(Stratagene, La Jolla, USA)にクローニングし、配列決定した。Vector NTIソフトウェア(Invitrogen)を使用して、結果をゲノムRab38配列と比較した。
Claims (14)
- ゲノム中に改変された標的配列を保有する非ヒト哺乳動物卵母細胞を作製する方法であって、該方法が、非ヒト哺乳動物卵母細胞に
(a) クラスター化され、規則的に間を開けられた短いパリンドロームリピート(CRISPR)関連タンパク質9(Cas9タンパク質)または前記Cas9をコードする核酸分子;ならびに
(b-i) 標的配列特異的CRISPR RNA (crRNA)およびトランス活性化crRNA (tracr RNA)もしくは前記RNAをコードする核酸分子;または
(b-ii) 標的配列特異的crRNAおよびtracrRNAを含むキメラRNA配列もしくは前記RNAをコードする核酸分子
を導入する工程を含み、
ここで、(a)において導入される該Cas9タンパク質および(b-i)または(b-ii)において導入されるRNA配列(1つまたは複数)は、標的配列に特異的に結合し、かつ標的配列中に一本鎖または二本鎖の切断(break)を導入するタンパク質/RNA複合体を形成する、方法。 - 標的配列が、ドナー核酸配列を用いて、相同組み換えにより改変され、
該方法が、
(c) 核酸分子を細胞に導入する工程をさらに含み、該核酸分子が、ドナー核酸配列および標的配列に相同な領域を含む、請求項1記載の方法。 - 核酸分子が一本鎖オリゴデオキシヌクレオチド(oligodesoxynucleotide)である、請求項2記載の方法。
- 卵母細胞が、受精した卵母細胞である、請求項1〜3いずれか記載の方法。
- Cas9タンパク質もしくは同タンパク質をコードする核酸分子および/または(b-i)もしくは(b-ii)のRNAまたは前記RNAをコードする核酸分子が、マイクロインジェクションにより卵母細胞に導入される、請求項1〜4いずれか記載の方法。
- (c)の核酸分子が、マイクロインジェクションにより卵母細胞に導入される、請求項2〜5いずれか記載の方法。
- Cas9タンパク質をコードする核酸分子がmRNAである、請求項1〜6いずれか記載の方法。
- Cas9タンパク質が配列番号:2に示されるアミノ酸配列を有する、請求項1〜7いずれか記載の方法。
- 標的配列に相同な領域が、ドナー核酸配列の5'末端および3'末端に位置する、請求項2〜8いずれか記載の方法。
- (c)の核酸分子に含まれる標的配列に相同な領域が、少なくとも400bpの長さを有する、請求項2〜9いずれか記載の方法。
- 標的配列の改変が、標的配列の少なくとも一ヌクレオチドの置換、挿入および欠失からなる群より選択される、請求項1〜10いずれか記載の方法。
- 卵母細胞が、げっ歯類、イヌ、ネコ、霊長類、ウサギ、ブタおよび反芻動物からなる群より選択される非ヒト哺乳動物由来である、請求項1〜11いずれか記載の方法。
- ゲノム中に改変された標的配列を保有する非ヒト哺乳動物を作製する方法であって、
(a) 請求項1〜12のいずれかに従って卵母細胞を作製する工程;
(b) (a)において得られた卵母細胞を偽妊娠雌宿主に移す工程;および
(c) 該雌宿主により出産された子孫を、改変の存在について分析する工程
を含む、方法。 - 該非ヒト哺乳動物が、げっ歯類、イヌ、ネコ、霊長類、ウサギ、ブタおよび反芻動物からなる群より選択される、請求項13記載の方法。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017170771A1 (ja) * | 2016-04-01 | 2017-10-05 | 国立大学法人徳島大学 | Cas9タンパク質を哺乳動物の受精卵に導入する方法 |
WO2018030208A1 (ja) * | 2016-08-10 | 2018-02-15 | 国立大学法人東京医科歯科大学 | 遺伝子ノックイン細胞の作製方法 |
CN112020560A (zh) * | 2018-04-25 | 2020-12-01 | 中国农业大学 | 一种RNA编辑的CRISPR/Cas效应蛋白及*** |
Families Citing this family (117)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2734621B1 (en) | 2011-07-22 | 2019-09-04 | President and Fellows of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
PL2847335T3 (pl) | 2012-04-25 | 2019-01-31 | Regeneron Pharmaceuticals, Inc. | Celowanie dużymi wektorami do celowania wspomagane nukleazą |
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 |
KR102210319B1 (ko) | 2013-03-15 | 2021-02-01 | 더 제너럴 하스피탈 코포레이션 | 특정 게놈 좌위에 대한 유전적 및 후성적 조절 단백질의 rna-안내 표적화 |
WO2014204578A1 (en) | 2013-06-21 | 2014-12-24 | The General Hospital Corporation | Using rna-guided foki nucleases (rfns) to increase specificity for rna-guided genome editing |
HUE040575T2 (hu) | 2013-04-16 | 2019-03-28 | Regeneron Pharma | A patkány genom célzott módosítása |
US9163284B2 (en) | 2013-08-09 | 2015-10-20 | President And Fellows Of Harvard College | Methods for identifying a target site of a Cas9 nuclease |
US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US9340799B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | MRNA-sensing switchable gRNAs |
US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
DE202014010413U1 (de) | 2013-09-18 | 2015-12-08 | Kymab Limited | Zellen und Organismen |
DK3066201T3 (en) | 2013-11-07 | 2018-06-06 | Editas Medicine Inc | CRISPR-RELATED PROCEDURES AND COMPOSITIONS WITH LEADING GRADES |
EP4349980A2 (en) | 2013-12-11 | 2024-04-10 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for the targeted modification of a genome |
MX2016007654A (es) | 2013-12-11 | 2017-08-15 | Regeneron Pharma | Metodos y composiciones para la modificacion dirigida de un genoma. |
US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
EP3080611B1 (en) | 2013-12-13 | 2018-11-14 | The General Hospital Corporation | Soluble high molecular weight (hmw) tau species and applications thereof |
LT3152312T (lt) | 2014-06-06 | 2020-04-27 | Regeneron Pharmaceuticals, Inc. | Tikslinio lokuso modifikavimo būdai ir kompozicijos |
HUE049405T2 (hu) | 2014-06-23 | 2020-09-28 | Regeneron Pharma | Nukleáz-közvetített DNS-összeállítás |
HUE041584T2 (hu) | 2014-06-26 | 2019-05-28 | Regeneron Pharma | Célzott genetikai módosítások és alkalmazási módszerek és készítmények |
US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
CN106922154B (zh) * | 2014-08-06 | 2022-01-07 | 基因工具股份有限公司 | 使用空肠弯曲杆菌crispr/cas***衍生的rna引导的工程化核酸酶的基因编辑 |
US10040048B1 (en) | 2014-09-25 | 2018-08-07 | Synthego Corporation | Automated modular system and method for production of biopolymers |
WO2016054032A1 (en) | 2014-09-29 | 2016-04-07 | The Jackson Laboratory | High efficiency, high throughput generation of genetically modified mammals by electroporation |
PL3207124T3 (pl) | 2014-10-15 | 2019-11-29 | Regeneron Pharma | Sposoby i kompozycje do wytwarzania lub utrzymywania komórek pluripotencjalnych |
US11470826B2 (en) | 2014-11-17 | 2022-10-18 | National University Corporation Tokyo Medical And Dental University | Method of conveniently producing genetically modified non-human mammal with high efficiency |
LT3221457T (lt) | 2014-11-21 | 2019-06-10 | Regeneron Pharmaceuticals, Inc. | Nukreipiančios genetinės modifikacijos būdai ir kompozicijos, naudojant suporuotas kreipiančiąsias rnr sekas |
GB201421096D0 (en) | 2014-11-27 | 2015-01-14 | Imp Innovations Ltd | Genome editing methods |
CN107208096B (zh) | 2014-12-18 | 2021-02-12 | 综合基因技术公司 | 基于crispr的组合物和使用方法 |
WO2016097751A1 (en) * | 2014-12-18 | 2016-06-23 | The University Of Bath | Method of cas9 mediated genome engineering |
RU2707137C2 (ru) | 2014-12-19 | 2019-11-22 | Регенерон Фармасьютикалз, Инк. | Способы и композиции для нацеленной генетической модификации посредством одноэтапного множественного нацеливания |
WO2016100857A1 (en) | 2014-12-19 | 2016-06-23 | Regeneron Pharmaceuticals, Inc. | Stem cells for modeling type 2 diabetes |
WO2016168763A1 (en) * | 2015-04-16 | 2016-10-20 | GenOva Laboratories LLC | Mitochondrial genome editing |
CA2993431A1 (en) | 2015-07-31 | 2017-02-09 | Regents Of The University Of Minnesota | Nuclease based knockouts of immunological checkpoint genes in immune cells |
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 |
WO2017040348A1 (en) | 2015-08-28 | 2017-03-09 | The General Hospital Corporation | Engineered crispr-cas9 nucleases |
JP7059179B2 (ja) * | 2015-10-20 | 2022-04-25 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | 遺伝子操作のための方法及び製品 |
IL310721A (en) | 2015-10-23 | 2024-04-01 | Harvard College | Nucleobase editors and their uses |
WO2017104404A1 (ja) * | 2015-12-18 | 2017-06-22 | 国立研究開発法人科学技術振興機構 | 遺伝子改変非ヒト生物、卵細胞、受精卵、及び標的遺伝子の改変方法 |
EP3402327A1 (en) * | 2016-01-15 | 2018-11-21 | The Jackson Laboratory | Genetically modified non-human mammals by multi-cycle electroporation of cas9 protein |
WO2017152023A1 (en) * | 2016-03-04 | 2017-09-08 | Indoor Biotechnologies Inc. | Fel d 1 knockouts and associated compositions and methods based on crispr-cas9 genomic editing |
EP3219799A1 (en) | 2016-03-17 | 2017-09-20 | IMBA-Institut für Molekulare Biotechnologie GmbH | Conditional crispr sgrna expression |
RU2745563C2 (ru) | 2016-05-20 | 2021-03-29 | Регенерон Фармасьютикалс, Инк. | Способы преодоления иммунологической толерантности с использованием множества направляющих рнк |
US11608570B2 (en) | 2016-07-29 | 2023-03-21 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Targeted in situ protein diversification by site directed DNA cleavage and repair |
KR20190041476A (ko) | 2016-07-29 | 2019-04-22 | 리제너론 파마슈티칼스 인코포레이티드 | C-절단된 피브릴린-1의 발현을 유도하는 돌연변이를 포함하는 마우스 |
CA3032699A1 (en) | 2016-08-03 | 2018-02-08 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
AU2017308889B2 (en) | 2016-08-09 | 2023-11-09 | President And Fellows Of Harvard College | Programmable Cas9-recombinase fusion proteins and uses thereof |
WO2018039438A1 (en) | 2016-08-24 | 2018-03-01 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
CN110214180A (zh) | 2016-10-14 | 2019-09-06 | 哈佛大学的校长及成员们 | 核碱基编辑器的aav递送 |
WO2018097257A1 (ja) | 2016-11-28 | 2018-05-31 | 国立大学法人大阪大学 | ゲノム編集方法 |
WO2018119359A1 (en) | 2016-12-23 | 2018-06-28 | President And Fellows Of Harvard College | Editing of ccr5 receptor gene to protect against hiv infection |
CA3049980A1 (en) | 2017-01-23 | 2018-07-26 | Regeneron Pharmaceuticals, Inc. | Hydroxysteroid 17-beta dehydrogenase 13 (hsd17b13) variants and uses thereof |
EP3592853A1 (en) | 2017-03-09 | 2020-01-15 | 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 |
CN110914426A (zh) | 2017-03-23 | 2020-03-24 | 哈佛大学的校长及成员们 | 包含核酸可编程dna结合蛋白的核碱基编辑器 |
WO2018195129A1 (en) | 2017-04-17 | 2018-10-25 | University Of Maryland, College Park | Embryonic cell cultures and methods of using the same |
CA3059956A1 (en) | 2017-04-21 | 2018-10-25 | The General Hospital Corporation | Variants of cpf1 (cas12a) with altered pam specificity |
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 |
SG11201911597YA (en) | 2017-06-05 | 2020-01-30 | Regeneron Pharma | B4galt1 variants and uses thereof |
CN111801345A (zh) | 2017-07-28 | 2020-10-20 | 哈佛大学的校长及成员们 | 使用噬菌体辅助连续进化(pace)的进化碱基编辑器的方法和组合物 |
AU2018309716A1 (en) | 2017-07-31 | 2020-01-16 | Regeneron Pharmaceuticals, Inc. | Cas-transgenic mouse embryonic stem cells and mice and uses thereof |
AU2018309714A1 (en) | 2017-07-31 | 2020-01-30 | Regeneron Pharmaceuticals, Inc. | Assessment of CRISPR/Cas-induced recombination with an exogenous donor nucleic acid in vivo |
CN111182790A (zh) | 2017-07-31 | 2020-05-19 | 瑞泽恩制药公司 | Crispr报告体非人类动物及其用途 |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
EP4276185A3 (en) | 2017-09-29 | 2024-02-21 | Regeneron Pharmaceuticals, Inc. | Rodents comprising a humanized ttr locus and methods of use |
AU2018352592A1 (en) | 2017-10-16 | 2020-06-04 | Beam Therapeutics, Inc. | Uses of adenosine base editors |
WO2019148166A1 (en) * | 2018-01-29 | 2019-08-01 | Massachusetts Institute Of Technology | Methods of enhancing chromosomal homologous recombination |
CA3089331A1 (en) | 2018-03-19 | 2019-09-26 | Regeneron Pharmaceuticals, Inc. | Transcription modulation in animals using crispr/cas systems |
EP3875469A4 (en) * | 2018-10-29 | 2022-08-17 | China Agricultural University | NEW CRISPR/CAS12F ENZYME AND SYSTEM |
CN113015799A (zh) | 2018-11-16 | 2021-06-22 | 国立大学法人大阪大学 | 经基因组编辑的细胞的制造方法 |
KR20200071198A (ko) | 2018-12-10 | 2020-06-19 | 네오이뮨텍, 인코퍼레이티드 | Nrf2 발현 조절 기반 T 세포 항암면역치료법 |
IL301193A (en) | 2018-12-20 | 2023-05-01 | Regeneron Pharma | Nuclease-mediated repeat expansion |
WO2020163396A1 (en) | 2019-02-04 | 2020-08-13 | The General Hospital Corporation | Adenine dna base editor variants with reduced off-target rna editing |
JP7461368B2 (ja) | 2019-03-18 | 2024-04-03 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | タウの播種または凝集の遺伝的修飾因子を同定するためのcrispr/casスクリーニングプラットフォーム |
JP7389135B2 (ja) | 2019-03-18 | 2023-11-29 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | タウ凝集に関連する遺伝的脆弱性を明らかにするためのcrispr/casドロップアウトスクリーニングプラットフォーム |
AU2020242032A1 (en) | 2019-03-19 | 2021-10-07 | Massachusetts Institute Of Technology | Methods and compositions for editing nucleotide sequences |
AU2020256225A1 (en) | 2019-04-03 | 2021-09-02 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for insertion of antibody coding sequences into a safe harbor locus |
IL286917B (en) | 2019-04-04 | 2022-09-01 | Regeneron Pharma | Methods for scar-free insertion of targeted modifications into targeted vectors |
IL286905B1 (en) | 2019-04-04 | 2024-02-01 | Regeneron Pharma | Non-human animals containing the human coagulation factor 12 locus |
EP3801011A1 (en) | 2019-06-04 | 2021-04-14 | Regeneron Pharmaceuticals, Inc. | Non-human animals comprising a humanized ttr locus with a beta-slip mutation and methods of use |
KR20220017939A (ko) | 2019-06-07 | 2022-02-14 | 리제너론 파마슈티칼스 인코포레이티드 | 인간화 알부민 좌위를 포함하는 비-인간 동물 |
AU2020290509A1 (en) | 2019-06-14 | 2021-11-11 | Regeneron Pharmaceuticals, Inc. | Models of tauopathy |
JP2022548031A (ja) | 2019-09-13 | 2022-11-16 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | 脂質ナノ粒子によって送達されるcrispr/casシステムを使用する動物における転写調節 |
KR20220097414A (ko) | 2019-11-08 | 2022-07-07 | 리제너론 파마슈티칼스 인코포레이티드 | X-연관 연소 망막층간분리 치료법을 위한 crispr 및 aav 전략 |
WO2021108363A1 (en) | 2019-11-25 | 2021-06-03 | Regeneron Pharmaceuticals, Inc. | Crispr/cas-mediated upregulation of humanized ttr allele |
WO2021144574A1 (en) | 2020-01-14 | 2021-07-22 | Pig Improvement Company Uk Limited | Gene editing of unfertilized porcine and bovine oocytes |
EP4114946A1 (en) | 2020-03-04 | 2023-01-11 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for sensitization of tumor cells to immune therapy |
US20230102342A1 (en) | 2020-03-23 | 2023-03-30 | Regeneron Pharmaceuticals, Inc. | Non-human animals comprising a humanized ttr locus comprising a v30m mutation and methods of use |
CA3177481A1 (en) | 2020-05-08 | 2021-11-11 | David R. Liu | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
WO2022050377A1 (ja) | 2020-09-04 | 2022-03-10 | 国立大学法人広島大学 | 標的dnaの編集方法、標的dnaが編集された細胞の製造方法、及びそれらに用いるdna編集システム |
WO2022120022A1 (en) | 2020-12-02 | 2022-06-09 | Regeneron Pharmaceuticals, Inc. | Crispr sam biosensor cell lines and methods of use thereof |
EP4337769A1 (en) | 2021-05-10 | 2024-03-20 | SQZ Biotechnologies Company | Methods for delivering genome editing molecules to the nucleus or cytosol of a cell and uses thereof |
WO2022251644A1 (en) | 2021-05-28 | 2022-12-01 | Lyell Immunopharma, Inc. | Nr4a3-deficient immune cells and uses thereof |
KR20240027676A (ko) | 2021-06-02 | 2024-03-04 | 라이엘 이뮤노파마, 인크. | Nr4a3-결핍 면역 세포 및 이의 용도 |
WO2023054573A1 (ja) | 2021-09-30 | 2023-04-06 | 国立大学法人大阪大学 | 相同染色体の一方に特異的なdna欠失を有する細胞を製造する方法 |
WO2023064924A1 (en) | 2021-10-14 | 2023-04-20 | Codiak Biosciences, Inc. | Modified producer cells for extracellular vesicle production |
WO2023077053A2 (en) | 2021-10-28 | 2023-05-04 | Regeneron Pharmaceuticals, Inc. | Crispr/cas-related methods and compositions for knocking out c5 |
CA3238939A1 (en) | 2021-12-08 | 2023-06-15 | Gaurang Patel | Mutant myocilin disease model and uses thereof |
US20230279442A1 (en) | 2021-12-15 | 2023-09-07 | Versitech Limited | Engineered cas9-nucleases and method of use thereof |
WO2023129974A1 (en) | 2021-12-29 | 2023-07-06 | Bristol-Myers Squibb Company | Generation of landing pad cell lines |
WO2023150181A1 (en) | 2022-02-01 | 2023-08-10 | President And Fellows Of Harvard College | Methods and compositions for treating cancer |
TW202332767A (zh) | 2022-02-02 | 2023-08-16 | 美商雷傑納榮製藥公司 | 用於治療龐貝氏症之抗TfR:GAA及抗CD63:GAA*** |
WO2023212677A2 (en) | 2022-04-29 | 2023-11-02 | Regeneron Pharmaceuticals, Inc. | Identification of tissue-specific extragenic safe harbors for gene therapy approaches |
WO2023220603A1 (en) | 2022-05-09 | 2023-11-16 | Regeneron Pharmaceuticals, Inc. | Vectors and methods for in vivo antibody production |
WO2023225665A1 (en) | 2022-05-19 | 2023-11-23 | Lyell Immunopharma, Inc. | Polynucleotides targeting nr4a3 and uses thereof |
WO2023235726A2 (en) | 2022-05-31 | 2023-12-07 | Regeneron Pharmaceuticals, Inc. | Crispr interference therapeutics for c9orf72 repeat expansion disease |
WO2023235725A2 (en) | 2022-05-31 | 2023-12-07 | Regeneron Pharmaceuticals, Inc. | Crispr-based therapeutics for c9orf72 repeat expansion disease |
WO2024026474A1 (en) | 2022-07-29 | 2024-02-01 | Regeneron Pharmaceuticals, Inc. | Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle |
WO2024031053A1 (en) | 2022-08-05 | 2024-02-08 | Regeneron Pharmaceuticals, Inc. | Aggregation-resistant variants of tdp-43 |
WO2024064958A1 (en) | 2022-09-23 | 2024-03-28 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells |
WO2024064952A1 (en) | 2022-09-23 | 2024-03-28 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells overexpressing c-jun |
WO2024073606A1 (en) | 2022-09-28 | 2024-04-04 | Regeneron Pharmaceuticals, Inc. | Antibody resistant modified receptors to enhance cell-based therapies |
WO2024077174A1 (en) | 2022-10-05 | 2024-04-11 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells |
WO2024098002A1 (en) | 2022-11-04 | 2024-05-10 | Regeneron Pharmaceuticals, Inc. | Calcium voltage-gated channel auxiliary subunit gamma 1 (cacng1) binding proteins and cacng1-mediated delivery to skeletal muscle |
WO2024107765A2 (en) | 2022-11-14 | 2024-05-23 | Regeneron Pharmaceuticals, Inc. | Compositions and methods for fibroblast growth factor receptor 3-mediated delivery to astrocytes |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011051390A1 (en) * | 2009-10-28 | 2011-05-05 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Homologous recombination in the oocyte |
WO2011154393A1 (en) * | 2010-06-07 | 2011-12-15 | 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 |
WO2013176772A1 (en) * | 2012-05-25 | 2013-11-28 | The Regents Of The University Of California | Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription |
WO2014089290A1 (en) * | 2012-12-06 | 2014-06-12 | Sigma-Aldrich Co. Llc | Crispr-based genome modification and regulation |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5389665B2 (ja) * | 2006-12-22 | 2014-01-15 | ジェネンテック, インコーポレイテッド | ヒト化vegfを発現するトランスジェニックマウス |
WO2011146121A1 (en) | 2010-05-17 | 2011-11-24 | Sangamo Biosciences, Inc. | Novel dna-binding proteins and uses thereof |
RU2014153918A (ru) * | 2012-06-12 | 2016-07-27 | Дженентек, Инк. | Способы и композиции для получения условно нокаутных аллелей |
WO2014001327A1 (en) | 2012-06-27 | 2014-01-03 | Telefonaktiebolaget Lm Ericsson (Publ) | Mobile device, network node, p2p-client, p2p-tracker and respective methods therein for performing a p2p-session |
GB201216564D0 (en) * | 2012-09-17 | 2012-10-31 | Univ Edinburgh | Genetically edited animal |
US20140273230A1 (en) | 2013-03-15 | 2014-09-18 | Sigma-Aldrich Co., Llc | Crispr-based genome modification and regulation |
-
2014
- 2014-02-27 JP JP2015558501A patent/JP2016507244A/ja active Pending
- 2014-02-27 EP EP14706855.5A patent/EP2922393B2/en active Active
- 2014-02-27 WO PCT/EP2014/053840 patent/WO2014131833A1/en active Application Filing
-
2015
- 2015-08-26 US US14/836,231 patent/US9783780B2/en active Active
-
2016
- 2016-07-13 US US15/209,516 patent/US10214723B2/en active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011051390A1 (en) * | 2009-10-28 | 2011-05-05 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) | Homologous recombination in the oocyte |
WO2011154393A1 (en) * | 2010-06-07 | 2011-12-15 | 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 |
WO2013176772A1 (en) * | 2012-05-25 | 2013-11-28 | The Regents Of The University Of California | Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription |
JP2015523856A (ja) * | 2012-05-25 | 2015-08-20 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフ | Rna依存性標的dna修飾およびrna依存性転写調節のための方法および組成物 |
WO2014089290A1 (en) * | 2012-12-06 | 2014-06-12 | Sigma-Aldrich Co. Llc | Crispr-based genome modification and regulation |
JP2016502840A (ja) * | 2012-12-06 | 2016-02-01 | 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Sigma−Aldrich Co., LLC | Crisprに基づくゲノム修飾および制御 |
Non-Patent Citations (5)
Title |
---|
L CONG: "MULTIPLEX GENOME ENGINEERING 以下省略", SCIENCE, vol. V339 N6121, JPN5016002455, 15 February 2013 (2013-02-15), pages 819 - 823, ISSN: 0003541959 * |
LAURENT TESSON, NATURE BIOTECHNOLOGY,2011年 8月5日,V29 N8,P695-696, JPN6018004162, ISSN: 0003733732 * |
MELANIE MEYER, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. V109 N24, JPN5016002453, 12 June 2012 (2012-06-12), US, pages 9354 - 9359, ISSN: 0003541958 * |
P MALI: "RNA-GUIDED HUMAN GENOME ENGINEERING VIA CAS9", SCIENCE, vol. V339 N6121, JPN5016002456, 3 January 2013 (2013-01-03), pages 823 - 826, ISSN: 0003541960 * |
WOONG Y HWANG, NATURE BIOTECHNOLOGY, vol. V31 N3, JPN5016002457, 29 January 2013 (2013-01-29), pages 227 - 229, ISSN: 0003541961 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017170771A1 (ja) * | 2016-04-01 | 2017-10-05 | 国立大学法人徳島大学 | Cas9タンパク質を哺乳動物の受精卵に導入する方法 |
WO2018030208A1 (ja) * | 2016-08-10 | 2018-02-15 | 国立大学法人東京医科歯科大学 | 遺伝子ノックイン細胞の作製方法 |
JPWO2018030208A1 (ja) * | 2016-08-10 | 2019-06-06 | 国立大学法人 東京医科歯科大学 | 遺伝子ノックイン細胞の作製方法 |
CN112020560A (zh) * | 2018-04-25 | 2020-12-01 | 中国农业大学 | 一种RNA编辑的CRISPR/Cas效应蛋白及*** |
CN112020560B (zh) * | 2018-04-25 | 2024-02-23 | 中国农业大学 | 一种RNA编辑的CRISPR/Cas效应蛋白及*** |
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US20150376652A1 (en) | 2015-12-31 |
US10214723B2 (en) | 2019-02-26 |
WO2014131833A1 (en) | 2014-09-04 |
US20160319242A1 (en) | 2016-11-03 |
EP2922393B2 (en) | 2022-12-28 |
EP2922393B1 (en) | 2019-09-04 |
EP2922393A1 (en) | 2015-09-30 |
US9783780B2 (en) | 2017-10-10 |
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