JP7201294B2 - 多重rna誘導型ゲノム編集 - Google Patents
多重rna誘導型ゲノム編集 Download PDFInfo
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Description
本願は、2013年7月9日に提出された米国仮特許出願第61/844168号に基づく優先権を主張するものであり、あらゆる目的のため、その全体が参照により本明細書に取り込まれる。
本発明は、米国エネルギー省助成金番号DE-FG02-02ER63445、全米科学財団助成金番号NSF-SynBERC、および全米科学財団助成金番号SA5283-11210の国庫助成により行われた。米国政府は本発明に関して一定の権利を有する。
酵母におけるCRISPR-Cas9を用いた多重遺伝子編集の一般的プロセス
ストレプトコッカス・ピオゲネス(Streptococcous pyogenes)のCRISPR免疫系由来のCas9を用いて、相同組換えを促進し、サッカロミセス・セレビシエ(Saccharaomyces cerevisiae)において形質転換DNAを組み換えない細胞以外を選択する。Cas9を用いたRNA誘導型DNA切断の一般的な方法を図1に示す。Cas9、ガイドRNA、および標的DNAの間で共局在複合体が形成される。Cas9により標的DNA中に二本鎖切断が生じる。次に、相同組換えによりドナーDNAがDNAに挿入される。ドナーDNAには、切断部位の両側に隣接配列、およびCas9切断部位を除去する配列が含まれる。その結果、ドナーDNAが、ゲノムDNAであってもよいDNA中に組み込まれる。
詳細な繰り返しプロトコール
(ウラシル栄養要求株、構成的Cas9発現)細胞を、5mlのSC酵母培地またはSC+FOA(100μg/ml)中で、光学密度0.8~1.0まで成長させる。細胞を2250×gで3分間スピンし、10mlの水で1回洗浄する。細胞をスピンし、1mlの100mM酢酸リチウムに再懸濁する。細胞をペレット化し、500μlの100mM酢酸リチウムに再懸濁する。50μlの細胞;1nmolの二本鎖オリゴヌクレオチドプール、各5μgのガイドRNA(ウラシルマーカーを有するp426ベクター)を含み、70μlまで水を加えて所望の最終体積にしたDNA混合物;240μlの50%PEG3350;および36μlの1M酢酸リチウムをこの順番で添加することにより、形質転換混合物を調製する。混合物を30℃で30分間インキュベートする。次に、混合物をボルテックスし、混合物を42℃で20分間インキュベートすることにより細胞に熱ショックを与える。次に、細胞をペレット化し、上清を除去する。細胞を5mlのSC-ウラシルに播種して、ウラシル遺伝子を含むgRNAプラスミドを選択する。細胞を2日間回復させる。2日後、100μlの細胞培養物を5mlの新たに調製したSCに播種し、12時間成長させてプラスミド維持について非選択とする。次に、100μlのSC培養細胞を5mlのSC+FOA(100μg/mL)培地に播種して、プラスミドを有する細胞以外を選択する。これにより、プロセスの1サイクルが完了する。このプロセスを、所望のサイクル回数分、反復する。プロセス全体は、1サイクル、2サイクル、3サイクル、4サイクル、5サイクル、6サイクル、7サイクル、8サイクル、9サイクル、10サイクル、15サイクル、20サイクル、25サイクルなどを含んでいてもよい。
選択された変異体における熱ショックに対する熱耐性
本明細書に記載の方法を用いて、選択された変異体における熱ショックに対する熱耐性が示された。ノックアウトまたは点突然変異により酵母の熱耐性を増大させることが示されている遺伝子を、本明細書に記載のガイドRNA-Cas9システムにより標的化した。変異について、4個の遺伝子、すなわちUBC1、SCH9、TFS1、およびRAS2を選択した。SCH9は、浸透圧ストレス(osmostress)、栄養素、および環境ストレスの遺伝子を制御するタンパク質キナーゼである。TFS1は、カルボキシペプチダーゼYおよびIra2pを阻害し、Ras GAP活性を阻害し、DNA複製ストレスに応答する。RAS2は、窒素飢餓を制御するGTP結合タンパク質であり、ストレス応答経路に関与する。SCH9、TFS1、およびRAS2のそれぞれについて、コード領域にセリンからアラニンへの変異を含むアレルであるドナーDNAを作成した。UBC1-E2はユビキチン結合酵素である。リン酸化部位を除去することで熱耐性を生じる点突然変異を含むドナーDNAを作成した。
本開示に係る態様を以下に例示する。
<1>
標的DNAに相補的なRNAと共局在複合体を形成、且つ前記標的DNAを部位特異的に切断する酵素を発現する細胞において前記標的DNAに複数の変化を生じさせる方法であって、
(a)標的DNAに相補的であり且つ前記酵素を前記標的DNAにガイドする、1種類または複数種類のRNAをコードする第1の外来核酸を前記細胞に導入すること、ここで、前記1種類または複数種類のRNAおよび前記酵素が、前記標的DNAへの共局在複合体の構成要素である、
1種類または複数種類のドナー核酸配列をコードする第2の外来核酸を前記細胞に導入すること、ここで、前記1種類または複数種類のRNAおよび前記1種類または複数種類のドナー核酸の配列が発現し、前記1種類または複数種類のRNAおよび前記酵素が前記標的DNAに共局在し、前記酵素により前記標的DNAが切断され、前記ドナー核酸が前記標的DNAに挿入されて前記細胞中に変化したDNAが生成する、および、
ステップ(a)を複数回繰り返して前記細胞において前記DNAに複数の変化を生じさせることを含む、方法。
<2>
前記酵素が、RNA誘導型DNA結合タンパク質である、<1>に記載の方法。
<3>
前記酵素がCas9である、<1>に記載の方法。
<4>
前記細胞が真核細胞である、<1>に記載の方法。
<5>
前記細胞が、酵母細胞、植物細胞、または動物細胞である、<1>に記載の方法。
<6>
前記RNAが、約10~約500ヌクレオチドである、<1>に記載の方法。
<7>
前記RNAが、約20~約100ヌクレオチドである、<1>に記載の方法。
<8>
前記1種類または複数種類のRNAが、ガイドRNAである、<1>に記載の方法。
<9>
前記1種類または複数種類のRNAが、tracrRNA-crRNA融合体である、<1>に記載の方法。
<10>
前記DNAが、ゲノムDNA、ミトコンドリアDNA、ウイルスDNA、または外来性DNAである、<1>に記載の方法。
<11>
前記1種類または複数種類のドナー核酸配列が、組換えにより挿入される、<1>に記載の方法。
<12>
前記1種類または複数種類のドナー核酸配列が、相同組換えにより挿入される、<1>に記載の方法。
<13>
前記1種類または複数種類のRNAおよび前記1種類または複数種類のドナー核酸配列が、1種類または複数種類のプラスミド上に存在する、<1>に記載の方法。
Claims (9)
- 標的DNAに相補的なガイドRNAと共局在複合体を形成し、且つ前記標的DNAを部位特異的に切断するCas9酵素を発現する細胞においてDNAに外来性核酸配列を多重挿入するインビトロ又はエクスビボの方法であって、
(a)複数種類のガイドRNAおよび複数種類の外来性ドナー核酸配列を前記細胞に導入すること、ここで、前記Cas9酵素により前記DNAが切断されて切断部位が形成され、該切断部位に前記複数種類の外来性ドナー核酸配列のうちの1つが挿入され、前記細胞は真核細胞である、および、
(b)ステップ(a)を繰り返して前記細胞において前記DNAに複数の外来性ドナー核酸配列挿入を生じさせることを含む、方法。 - 前記細胞が、酵母細胞、植物細胞、または動物細胞である、請求項1に記載の方法。
- 前記ガイドRNAが、tracrRNA-crRNA融合体である、請求項1に記載の方法。
- 前記標的DNAが、ゲノムDNA、ミトコンドリアDNA、ウイルスDNA、または外来性DNAである、請求項1に記載の方法。
- 前記外来性ドナー核酸配列が、組換えにより挿入される、請求項1に記載の方法。
- 前記外来性ドナー核酸配列が、相同組換えにより挿入される、請求項1に記載の方法。
- 前記複数種類のガイドRNA及び前記複数種類の外来性ドナー核酸配列が、1つ又は複数のプラスミド上に存在する、請求項1に記載の方法。
- 前記外来性ドナー核酸配列が前記切断部位の両側に隣接する相同配列またはアームを含む、請求項1に記載の方法。
- 前記外来性ドナー核酸配列が前記切断部位を除去するための配列を含む、請求項1に記載の方法。
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Families Citing this family (110)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2853829C (en) | 2011-07-22 | 2023-09-26 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
KR20150095861A (ko) * | 2012-12-17 | 2015-08-21 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | Rna-가이드된 인간 게놈 조작 |
EP2946015B1 (en) | 2013-01-16 | 2021-05-26 | Emory University | Cas9-nucleic acid complexes and uses related thereto |
SG11201508028QA (en) | 2013-04-16 | 2015-10-29 | Regeneron Pharma | Targeted modification of rat genome |
US20150044192A1 (en) | 2013-08-09 | 2015-02-12 | 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 |
US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
US9340800B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | Extended DNA-sensing GRNAS |
TW201542816A (zh) | 2013-09-18 | 2015-11-16 | Kymab Ltd | 方法、細胞與生物體 |
KR102380245B1 (ko) | 2013-11-07 | 2022-03-30 | 에디타스 메디신, 인코포레이티드 | 지배적인 gRNA를 이용하는 CRISPR-관련 방법 및 조성물 |
CN105980568B (zh) | 2013-12-11 | 2019-12-03 | 瑞泽恩制药公司 | 用于靶向修饰基因组的方法和组合物 |
US20150166982A1 (en) | 2013-12-12 | 2015-06-18 | President And Fellows Of Harvard College | Methods for correcting pi3k point mutations |
KR102496984B1 (ko) | 2014-02-11 | 2023-02-06 | 더 리전츠 오브 더 유니버시티 오브 콜로라도, 어 바디 코퍼레이트 | Crispr 이용의 다중화된 게놈 조작 |
WO2015134812A1 (en) | 2014-03-05 | 2015-09-11 | 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) |
US11141493B2 (en) | 2014-03-10 | 2021-10-12 | Editas Medicine, Inc. | Compositions and methods for treating CEP290-associated disease |
US11339437B2 (en) | 2014-03-10 | 2022-05-24 | Editas Medicine, Inc. | Compositions and methods for treating CEP290-associated disease |
EP3981876A1 (en) | 2014-03-26 | 2022-04-13 | Editas Medicine, Inc. | Crispr/cas-related methods and compositions for treating sickle cell disease |
WO2015184268A1 (en) | 2014-05-30 | 2015-12-03 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions and methods of delivering treatments for latent viral infections |
US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
WO2016073990A2 (en) | 2014-11-07 | 2016-05-12 | Editas Medicine, Inc. | Methods for improving crispr/cas-mediated genome-editing |
US10900034B2 (en) | 2014-12-03 | 2021-01-26 | Agilent Technologies, Inc. | Guide RNA with chemical modifications |
WO2016123514A1 (en) * | 2015-01-29 | 2016-08-04 | University Of Massachusetts | Nanoparticle-protein complex for intracellular protein delivery |
AU2016232819B2 (en) | 2015-03-19 | 2021-10-21 | Board Of Regents Of The University Of Texas System | Compositions and methods for use of anion channel rhodopsins |
EP3280803B1 (en) | 2015-04-06 | 2021-05-26 | The Board of Trustees of the Leland Stanford Junior University | Chemically modified guide rnas for crispr/cas-mediated gene regulation |
EP3286571B1 (en) | 2015-04-24 | 2021-08-18 | Editas Medicine, Inc. | Evaluation of cas9 molecule/guide rna molecule complexes |
EP3289080B1 (en) * | 2015-04-30 | 2021-08-25 | The Trustees of Columbia University in the City of New York | Gene therapy for autosomal dominant diseases |
US20160346359A1 (en) * | 2015-05-01 | 2016-12-01 | Spark Therapeutics, Inc. | Adeno-associated Virus-Mediated CRISPR-Cas9 Treatment of Ocular Disease |
CA2986310A1 (en) | 2015-05-11 | 2016-11-17 | Editas Medicine, Inc. | Optimized crispr/cas9 systems and methods for gene editing in stem cells |
US10117911B2 (en) | 2015-05-29 | 2018-11-06 | Agenovir Corporation | Compositions and methods to treat herpes simplex virus infections |
JP7396783B2 (ja) | 2015-06-09 | 2023-12-12 | エディタス・メディシン、インコーポレイテッド | 移植を改善するためのcrispr/cas関連方法および組成物 |
CA2999500A1 (en) | 2015-09-24 | 2017-03-30 | Editas Medicine, Inc. | Use of exonucleases to improve crispr/cas-mediated genome editing |
EP4269577A3 (en) | 2015-10-23 | 2024-01-17 | President and Fellows of Harvard College | Nucleobase editors and uses thereof |
EP4036228A1 (en) | 2015-11-13 | 2022-08-03 | Avellino Lab USA, Inc. | Methods for the treatment of corneal dystrophies |
WO2017165826A1 (en) | 2016-03-25 | 2017-09-28 | Editas Medicine, Inc. | Genome editing systems comprising repair-modulating enzyme molecules and methods of their use |
US11512311B2 (en) | 2016-03-25 | 2022-11-29 | Editas Medicine, Inc. | Systems and methods for treating alpha 1-antitrypsin (A1AT) deficiency |
EP4047092A1 (en) | 2016-04-13 | 2022-08-24 | Editas Medicine, Inc. | Cas9 fusion molecules, gene editing systems, and methods of use thereof |
US10767175B2 (en) | 2016-06-08 | 2020-09-08 | Agilent Technologies, Inc. | High specificity genome editing using chemically modified guide RNAs |
US11293021B1 (en) | 2016-06-23 | 2022-04-05 | Inscripta, Inc. | Automated cell processing methods, modules, instruments, and systems |
US10017760B2 (en) | 2016-06-24 | 2018-07-10 | Inscripta, Inc. | Methods for generating barcoded combinatorial libraries |
US11359234B2 (en) | 2016-07-01 | 2022-06-14 | Microsoft Technology Licensing, Llc | Barcoding sequences for identification of gene expression |
US10892034B2 (en) | 2016-07-01 | 2021-01-12 | Microsoft Technology Licensing, Llc | Use of homology direct repair to record timing of a molecular event |
WO2018005117A1 (en) | 2016-07-01 | 2018-01-04 | Microsoft Technology Licensing, Llc | Storage through iterative dna editing |
WO2018015995A1 (ja) * | 2016-07-19 | 2018-01-25 | 株式会社バイオダイナミクス研究所 | 長鎖一本鎖dnaを調製する方法 |
EP3494220A1 (en) | 2016-08-02 | 2019-06-12 | Editas Medicine, Inc. | Compositions and methods for treating cep290 associated disease |
SG11201900907YA (en) | 2016-08-03 | 2019-02-27 | Harvard College | Adenosine nucleobase editors and uses thereof |
WO2018031683A1 (en) | 2016-08-09 | 2018-02-15 | President And Fellows Of Harvard College | Programmable cas9-recombinase fusion proteins and uses thereof |
CN109963945A (zh) * | 2016-08-20 | 2019-07-02 | 阿维利诺美国实验室股份有限公司 | 单一向导rna、crispr/cas9系统及其使用方法 |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | 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 |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
JP2020505074A (ja) * | 2017-01-30 | 2020-02-20 | カー・ヴェー・エス ザート エス・エー ウント コー. カー・ゲー・アー・アーKWS SAAT SE & Co. KGaA | ゲノム工学のためのエンドヌクレアーゼに対する修復鋳型の結合 |
EP3592853A1 (en) | 2017-03-09 | 2020-01-15 | President and Fellows of Harvard College | Suppression of pain by gene editing |
EP3592777A1 (en) | 2017-03-10 | 2020-01-15 | 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 |
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 |
WO2018201086A1 (en) | 2017-04-28 | 2018-11-01 | 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 |
CN110997908A (zh) | 2017-06-09 | 2020-04-10 | 爱迪塔斯医药公司 | 工程化的cas9核酸酶 |
US10011849B1 (en) | 2017-06-23 | 2018-07-03 | Inscripta, Inc. | Nucleic acid-guided nucleases |
US9982279B1 (en) | 2017-06-23 | 2018-05-29 | Inscripta, Inc. | Nucleic acid-guided nucleases |
SI3645719T1 (sl) | 2017-06-30 | 2022-07-29 | Inscripta, Inc., | Postopki, moduli, instrumenti in sistemi za avtomatizirano obdelavo celic |
EP3652312A1 (en) | 2017-07-14 | 2020-05-20 | Editas Medicine, Inc. | Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites |
WO2019023680A1 (en) | 2017-07-28 | 2019-01-31 | President And Fellows Of Harvard College | METHODS AND COMPOSITIONS FOR EVOLUTION OF BASIC EDITORS USING PHAGE-ASSISTED CONTINUOUS EVOLUTION (PACE) |
RU2020111575A (ru) | 2017-08-22 | 2021-09-23 | Напиджен, Инк. | Модификация генома органелл с использованием направляемой полинуклеотидом эндонуклеазы |
US10738327B2 (en) | 2017-08-28 | 2020-08-11 | Inscripta, Inc. | Electroporation cuvettes for automation |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US10443074B2 (en) | 2017-09-30 | 2019-10-15 | Inscripta, Inc. | Modification of cells by introduction of exogenous material |
JP2021500036A (ja) | 2017-10-16 | 2021-01-07 | ザ ブロード インスティテュート, インコーポレーテッドThe Broad Institute, Inc. | アデノシン塩基編集因子の使用 |
CA3087715A1 (en) | 2018-02-08 | 2019-08-15 | Zymergen Inc. | Genome editing using crispr in corynebacterium |
CN112204131A (zh) | 2018-03-29 | 2021-01-08 | 因思科瑞普特公司 | 用于诱导和转化的细胞生长速率的自动化控制 |
WO2019200004A1 (en) | 2018-04-13 | 2019-10-17 | Inscripta, Inc. | Automated cell processing instruments comprising reagent cartridges |
US10858761B2 (en) | 2018-04-24 | 2020-12-08 | Inscripta, Inc. | Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells |
US10508273B2 (en) | 2018-04-24 | 2019-12-17 | Inscripta, Inc. | Methods for identifying selective binding pairs |
US10557216B2 (en) | 2018-04-24 | 2020-02-11 | Inscripta, Inc. | Automated instrumentation for production of T-cell receptor peptide libraries |
CN112654710A (zh) | 2018-05-16 | 2021-04-13 | 辛瑟高公司 | 用于指导rna设计和使用的方法和系统 |
CN114854720A (zh) | 2018-06-30 | 2022-08-05 | 因思科瑞普特公司 | 用于改进活细胞中编辑序列的检测的仪器、模块和方法 |
US11142740B2 (en) | 2018-08-14 | 2021-10-12 | Inscripta, Inc. | Detection of nuclease edited sequences in automated modules and instruments |
US10752874B2 (en) | 2018-08-14 | 2020-08-25 | Inscripta, Inc. | Instruments, modules, and methods for improved detection of edited sequences in live cells |
US10532324B1 (en) | 2018-08-14 | 2020-01-14 | Inscripta, Inc. | Instruments, modules, and methods for improved detection of edited sequences in live cells |
AU2019363487A1 (en) | 2018-08-30 | 2021-04-15 | Inscripta, Inc. | Improved detection of nuclease edited sequences in automated modules and instruments |
US10604746B1 (en) | 2018-10-22 | 2020-03-31 | Inscripta, Inc. | Engineered enzymes |
US11214781B2 (en) | 2018-10-22 | 2022-01-04 | Inscripta, Inc. | Engineered enzyme |
US11053515B2 (en) | 2019-03-08 | 2021-07-06 | Zymergen Inc. | Pooled genome editing in microbes |
JP2022524043A (ja) | 2019-03-08 | 2022-04-27 | ザイマージェン インコーポレイテッド | 微生物の反復ゲノム編集 |
JP2022524044A (ja) * | 2019-03-08 | 2022-04-27 | ザイマージェン インコーポレイテッド | 微生物のプール型ゲノム編集 |
BR112021018607A2 (pt) | 2019-03-19 | 2021-11-23 | Massachusetts Inst Technology | Métodos e composições para editar sequências de nucleotídeos |
US10815467B2 (en) | 2019-03-25 | 2020-10-27 | Inscripta, Inc. | Simultaneous multiplex genome editing in yeast |
US11001831B2 (en) | 2019-03-25 | 2021-05-11 | Inscripta, Inc. | Simultaneous multiplex genome editing in yeast |
WO2020247587A1 (en) | 2019-06-06 | 2020-12-10 | Inscripta, Inc. | Curing for recursive nucleic acid-guided cell editing |
US10907125B2 (en) | 2019-06-20 | 2021-02-02 | Inscripta, Inc. | Flow through electroporation modules and instrumentation |
US10920189B2 (en) | 2019-06-21 | 2021-02-16 | Inscripta, Inc. | Genome-wide rationally-designed mutations leading to enhanced lysine production in E. coli |
US10927385B2 (en) | 2019-06-25 | 2021-02-23 | Inscripta, Inc. | Increased nucleic-acid guided cell editing in yeast |
WO2021102059A1 (en) | 2019-11-19 | 2021-05-27 | Inscripta, Inc. | Methods for increasing observed editing in bacteria |
KR20220110778A (ko) | 2019-12-10 | 2022-08-09 | 인스크립타 인코포레이티드 | 신규 mad 뉴클레아제 |
US10704033B1 (en) | 2019-12-13 | 2020-07-07 | Inscripta, Inc. | Nucleic acid-guided nucleases |
JP2023507566A (ja) | 2019-12-18 | 2023-02-24 | インスクリプタ, インコーポレイテッド | 核酸誘導ヌクレアーゼ編集済み細胞のin vivo検出のためのカスケード/dCas3相補性アッセイ |
US10689669B1 (en) | 2020-01-11 | 2020-06-23 | Inscripta, Inc. | Automated multi-module cell processing methods, instruments, and systems |
KR20220133257A (ko) | 2020-01-27 | 2022-10-04 | 인스크립타 인코포레이티드 | 전기천공 모듈 및 기구 |
US20210332388A1 (en) | 2020-04-24 | 2021-10-28 | Inscripta, Inc. | Compositions, methods, modules and instruments for automated nucleic acid-guided nuclease editing in mammalian cells |
CN116096873A (zh) | 2020-05-08 | 2023-05-09 | 布罗德研究所股份有限公司 | 同时编辑靶标双链核苷酸序列的两条链的方法和组合物 |
US11787841B2 (en) | 2020-05-19 | 2023-10-17 | Inscripta, Inc. | Rationally-designed mutations to the thrA gene for enhanced lysine production in E. coli |
US11299731B1 (en) | 2020-09-15 | 2022-04-12 | Inscripta, Inc. | CRISPR editing to embed nucleic acid landing pads into genomes of live cells |
US11512297B2 (en) | 2020-11-09 | 2022-11-29 | Inscripta, Inc. | Affinity tag for recombination protein recruitment |
EP4271802A1 (en) | 2021-01-04 | 2023-11-08 | Inscripta, Inc. | Mad nucleases |
EP4274890A1 (en) | 2021-01-07 | 2023-11-15 | Inscripta, Inc. | Mad nucleases |
US11884924B2 (en) | 2021-02-16 | 2024-01-30 | Inscripta, Inc. | Dual strand nucleic acid-guided nickase editing |
AU2022343300A1 (en) | 2021-09-10 | 2024-04-18 | Agilent Technologies, Inc. | Guide rnas with chemical modification for prime editing |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010131014A (ja) | 1998-07-15 | 2010-06-17 | Maxygen Inc | 繰返し配列組換えによる細胞全体および生物の進化 |
US20130052646A1 (en) | 2009-08-10 | 2013-02-28 | Mascoma Corporation | Positive and negative selectable markers for use in thermophilic organisms |
JP2013520989A (ja) | 2010-03-05 | 2013-06-10 | シンセティック ジェノミクス インコーポレーテッド | ゲノムのクローニングおよび操作のための方法 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4935357A (en) | 1986-02-05 | 1990-06-19 | New England Biolabs, Inc. | Universal restriction endonuclease |
AU3391900A (en) * | 1999-03-05 | 2000-09-21 | Maxygen, Inc. | Encryption of traits using split gene sequences |
JP2005500841A (ja) * | 2001-07-27 | 2005-01-13 | アメリカ合衆国 | オリゴヌクレオチドを用いるインビボ部位指定変異誘発のためのシステム |
US20100076057A1 (en) * | 2008-09-23 | 2010-03-25 | Northwestern University | TARGET DNA INTERFERENCE WITH crRNA |
CN102858985A (zh) * | 2009-07-24 | 2013-01-02 | 西格马-奥尔德里奇有限责任公司 | 基因组编辑方法 |
KR101754083B1 (ko) * | 2009-09-25 | 2017-07-05 | 바스프 플랜트 사이언스 컴퍼니 게엠베하 | 향상된 수확량 관련 형질을 갖는 식물 및 이의 제조 방법 |
MX362866B (es) * | 2012-05-25 | 2019-02-20 | Univ California | Metodos y composiciones para la modificacion de adn objetivo dirigida por arn y para la modulacion de la transcripcion dirigida por arn. |
EP3372679A1 (en) * | 2012-10-23 | 2018-09-12 | Toolgen Incorporated | Composition for cleaving a target dna comprising a guide rna specific for the target dna and cas protein-encoding nucleic acid or cas protein, and use thereof |
WO2014093701A1 (en) * | 2012-12-12 | 2014-06-19 | The Broad Institute, Inc. | Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof |
CA2908512C (en) * | 2013-04-05 | 2023-10-24 | Dow Agrosciences Llc | Methods and compositions for integration of an exogenous sequence within the genome of plants |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010131014A (ja) | 1998-07-15 | 2010-06-17 | Maxygen Inc | 繰返し配列組換えによる細胞全体および生物の進化 |
US20130052646A1 (en) | 2009-08-10 | 2013-02-28 | Mascoma Corporation | Positive and negative selectable markers for use in thermophilic organisms |
JP2013520989A (ja) | 2010-03-05 | 2013-06-10 | シンセティック ジェノミクス インコーポレーテッド | ゲノムのクローニングおよび操作のための方法 |
Non-Patent Citations (6)
Title |
---|
Cell, 2013年5月9日, vol.153, pp.910-918 |
Nature Biotechnol.,2013年01月29日,Vol.31,pp.233-241 |
Nature,2009年,Vol.460,pp.894-898,METHODS |
Nucleic Acids Res.,2013年03月04日,Vol.41, No.7,pp.4336-4343 |
Science,2013年02月15日,Vol.339,pp.823-826, Supplementary Materials |
Science,Vol.339,2013年02月15日,pp.819-823, Supplementary Materials |
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