JP6839082B2 - 環状ポリヌクレオチド修飾鋳型と組み合わせてガイドrna/casエンドヌクレアーゼ系を用いるe.コリ(e.coli)での効率的な遺伝子編集のための組成物および方法 - Google Patents
環状ポリヌクレオチド修飾鋳型と組み合わせてガイドrna/casエンドヌクレアーゼ系を用いるe.コリ(e.coli)での効率的な遺伝子編集のための組成物および方法 Download PDFInfo
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Description
配列表の認証謄本が、2015年11月17日作成の20151117_CL6256PCT_ST25.txtのファイル名の、106キロバイトのサイズを有するASCIIフォーマットの配列表としてEFS−Webを介して電子的に提出され、明細書と同時に出願される。このASCIIフォーマットの文書内に含有される配列表は、本明細書の一部であり、参照によってその全体が本明細書に組み込まれる。
1.以下の小さい脂肪族の非極性または僅かな極性の残基は、互いに置換してよい:Ala(A)、Ser(S)、Thr(T)、Pro(P)、Gly(G);
2.以下の負に帯電した極性残基およびそれらのアミドは、互いに置換してよい:Asp(D)、Asn(N)、Glu(E)、Gln(Q);
3.以下の正に帯電した極性残基は、互いに置換してよい:His(H)、Arg(R)、Lys(K);
4.以下の脂肪族の非極性残基は、互いに置換してよい:Ala(A)、Leu(L)、Ile(I)、Val(V)、Cys(C)、Met(M);および
5.以下の大きい芳香族残基は、互いに置換してよい:Phe(F)、Tyr(Y)、Trp(W)。
Escherichia coli K−12 using PCR products.Proceedings of the National Academy of Sciences of the United States of America 97:6640−6645)。
1.エケリキア・コリ(Escherichia coli)細胞のゲノムのヌクレオチド配列を編集するための方法であって、ガイドRNAをコードするDNA配列および環状ポリヌクレオチド修飾鋳型を含む少なくとも1つの組換えDNAコンストラクトを、誘導プロモータに機能可能なように結合したCas9エンドヌクレアーゼDNA配列を含むE.コリ(E.coli)細胞に提供する工程を含み、前記Cas9エンドヌクレアーゼDNA配列が、前記E.コリ(E.coli)細胞のゲノムの標的部位に二重鎖切断を導入することができるCas9エンドヌクレアーゼをコードし、前記ポリヌクレオチド修飾鋳型が、前記ヌクレオチド配列の少なくとも1つのヌクレオチド修飾を含む、方法。
2.E.コリ(E.coli)細胞のゲノムのヌクレオチド配列が、プロモータ配列、ターミネータ配列、調節要素配列、コード配列、プロファージ、偽遺伝子、外来遺伝子、および内在性遺伝子からなる群から選択される、実施形態1に記載の方法。
3.ガイドRNAをコードするDNA配列を含む前記組換えDNAコンストラクトが、環状プラスミドによって提供される、実施形態1に記載の方法。
4.組換えDNAコンストラクトおよび環状ポリヌクレオチド修飾鋳型がそれぞれ、別個のプラスミドに提供される、実施形態1に記載の方法。
5.組換えDNAコンストラクトおよび環状ポリヌクレオチド修飾鋳型が、1つのプラスミドに提供される、実施形態1に記載の方法。
6.組換えDNAコンストラクトおよび環状ポリヌクレオチド鋳型が、エレクトロポレーション、熱ショック、ファージ送達、交配、コンジュゲーション、および形質導入からなる群から選択される1つの手段によって提供される、実施形態1に記載の方法。
7.前記標的部位が、第1のゲノム領域および第2のゲノム領域に隣接し、環状ポリヌクレオチド鋳型が、前記第1のゲノム領域に相同の第1の領域および前記第2のゲノム領域に相同の第2の領域をさらに含む、実施形態1に記載の方法。
8.E.コリ(E.coli)細胞が、外来組換えタンパク質を発現しない、実施形態1に記載の方法。
9.E.コリ(E.coli)細胞が、RecETタンパク質、λ−redタンパク質、およびRecBCD阻害剤を含む群から選択されるタンパク質を発現しない、実施形態1に記載の方法。
10.前記E.コリ(E.coli)細胞から子孫細胞を増殖させる工程をさらに含み、この子孫細胞が、前記ヌクレオチド配列の少なくとも1つのヌクレオチド修飾を含む、実施形態1に記載の方法。
11.標的部位が、E.コリ(E.coli)galK遺伝子内に位置する、実施形態1に記載の方法。
12.実施形態1に記載の方法によって作製されたE.コリ(E.coli)細胞。
13.実施形態12に記載のE.コリ(E.coli)細胞から作製されたE.コリ(E.coli)株。
14.galK変異E.コリ(E.coli)細胞を作製するための方法であって:
(a)ガイドRNAをコードするDNA配列および少なくとも1つの環状ポリヌクレオチド修飾鋳型を含む少なくとも1つの環状組換えDNAコンストラクトを、誘導プロモータに機能可能なように結合したCas9エンドヌクレアーゼDNA配列を含むE.コリ(E.coli)細胞に提供する工程であって、前記Cas9エンドヌクレアーゼDNA配列が、E.コリ(E.coli)ゲノムのgalKゲノム配列内の標的部位に二重鎖切断を導入することができるCasエンドヌクレアーゼをコードし、前記環状ポリヌクレオチド修飾鋳型が、前記galKゲノム配列の少なくとも1つのヌクレオチド修飾を含む、工程;
(b)(a)のE.コリ(E.coli)細胞から子孫細胞を増殖させる工程;および
(c)前記少なくとも1つのヌクレオチド修飾の存在について(b)の子孫細胞を評価する工程を含む、方法。
15.エケリキア・コリ(Escherichia coli)細胞のゲノムのヌクレオチド配列を編集するための方法であって、ガイドRNAをコードするDNA配列、環状ポリヌクレオチド修飾鋳型を含む少なくとも第1の組換えDNAコンストラクト、および誘導プロモータに機能可能なように結合したCas9エンドヌクレアーゼをコードするDNA配列を含む第2の組換えDNAコンストラクトをE.コリ(E.coli)細胞に提供する工程を含み、Cas9エンドヌクレアーゼが、前記E.コリ(E.coli)細胞のゲノムの標的部位に二重鎖切断を導入し、前記ポリヌクレオチド修飾鋳型が、前記ヌクレオチド配列の少なくとも1つのヌクレオチド修飾を含む、方法。
16.第1の組換えDNAコンストラクト、第2の組換えDNAコンストラクト、および環状ポリヌクレオチド修飾鋳型がそれぞれ、別個のプラスミドに提供される、実施形態15に記載の方法。
17.第1の組換えDNAコンストラクト、第2の組換えDNAコンストラクト、および環状ポリヌクレオチド修飾鋳型が、1つのプラスミドに提供される、実施形態1に記載の方法。
エケリキア・コリ(Escherichia coli)に使用されるCas9エンドヌクレアーゼ発現ベクターの作製
この実施例では、エケリキア・コリ(Escherichia coli)のゲノム編集用の誘導Cas9発現ベクターを作製した。インデューサ―に応答するCas9発現を確認した。
E.コリ(E.coli)のgalK遺伝子をターゲティングする単一ガイドRNAをコードする環状発現プラスミドの作製
E.コリ(E.coli)の内在性galK遺伝子を修飾(編集)するために、E.コリ(E.coli)galK遺伝子内の4つの(4)Cas9エンドヌクレアーゼ標的部位を特定した(図5):galK−1(配列番号13、表1)、galK−2(配列番号14、表1)、galK−3(配列番号15、表1)、およびgalK−4(配列番号16、表1)。
E.コリ(E.coli)での遺伝子編集のためのポリヌクレオチド修飾鋳型を含む環状プラスミドの作製
(例えば、galK遺伝子の遺伝子欠失)を用いるE.コリ(E.coli)での遺伝子編集(修飾)を可能にするために、以下のようにgalK遺伝子の一部が欠如したポリヌクレオチド修飾鋳型(galK欠失鋳型とも呼ばれる)を作製した:
ポリヌクレオチド修飾鋳型を含む環状プラスミドと組み合わせてガイドRNA/Casエンドヌクレアーゼ系を用いるE.コリ(E.coli)のgalK遺伝子の効率的なゲノム編集
E.コリ(E.coli)のgalE遺伝子の欠失を含む菌株EF44は、毒性産物リン−ガラクトースの蓄積により、増殖培地中のガラクトースの存在に感受性がある(Incorporate E.coli and S.typhimurium:Cellular and Molecular Biology Authors:Frederick C.Neidhardt,John L.Ingraham,Roy Curtiss III.ASM Press Washington D.C.1987)。この菌株では、ガラクトースキナーゼ(galK)をコードする遺伝子の機能の喪失をもたらす変異が、ガラクトース感受性を低下させ、菌株がガラクトースの存在下でも増殖することができる。
Claims (14)
- 外来組換えタンパク質を発現しないエケリキア・コリ(Escherichia coli:E.コリ(E.coli))細胞のゲノムの標的配列を編集するための方法であって、少なくとも環状ポリヌクレオチド修飾鋳型、および、ガイドRNAをコードするDNA配列に機能可能なように結合したプロモータを含む組換えDNAコンストラクトを、E.コリ(E.coli)細胞に提供する工程を含み、前記E.コリ(E.coli)細胞が、誘導プロモータに機能可能なように結合したCas9エンドヌクレアーゼDNA配列を含み、前記Cas9エンドヌクレアーゼDNA配列が、Cas9エンドヌクレアーゼをコードし、前記ガイドRNAおよびCas9エンドヌクレアーゼが、前記標的配列を切断することができるRNA誘導型エンドヌクレアーゼ(RGEN)を形成することができ、前記ポリヌクレオチド修飾鋳型が、前記標的配列の少なくとも1つのヌクレオチド修飾を含み、前記RGENは、前記環状ポリヌクレオチド修飾鋳型と共に、前記標的配列に前記少なくとも1つのヌクレオチド修飾を導入する、方法。
- E.コリ(E.coli)細胞のゲノムの標的配列が、プロモータ配列、ターミネータ配列、調節要素配列、コード配列、プロファージ、偽遺伝子、および外来遺伝子からなる群から選択される、請求項1に記載の方法。
- 前記組換えDNAコンストラクトが、環状プラスミドによって提供される、請求項1に記載の方法。
- 前記組換えDNAコンストラクトおよび前記環状ポリヌクレオチド修飾鋳型がそれぞれ、別個のプラスミドに提供される、請求項1に記載の方法。
- 前記組換えDNAコンストラクトおよび前記環状ポリヌクレオチド修飾鋳型が、1つのプラスミドに提供される、請求項1に記載の方法。
- 前記組換えDNAコンストラクトおよび前記環状ポリヌクレオチド修飾鋳型が、エレクトロポレーション、熱ショック、ファージ送達、交配、コンジュゲーション、および形質導入からなる群から選択される1つの手段によって提供される、請求項1に記載の方法。
- 前記標的部位が、第1のゲノム領域および第2のゲノム領域に隣接し、前記環状ポリヌクレオチド修飾鋳型が、前記第1のゲノム領域に相同の第1の領域および前記第2のゲノム領域に相同の第2の領域をさらに含む、請求項1に記載の方法。
- 前記E.コリ(E.coli)細胞が、RecETタンパク質、λ−redタンパク質、およびRecBCD阻害剤を含む群から選択されるタンパク質を発現しない、請求項1に記載の方法。
- 前記E.コリ(E.coli)細胞から子孫細胞を増殖させる工程をさらに含み、前記子孫細胞が、前記標的配列の少なくとも1つのヌクレオチド修飾を含む、請求項1に記載の方法。
- 標的配列が、E.コリ(E.coli)galK遺伝子内に位置する、請求項1に記載の方法。
- galK変異E.コリ(E.coli)細胞を作製するための方法であって:
a)ガイドRNAをコードするDNA配列に機能可能なように結合したプロモータを含む少なくとも1つの環状組換えDNAコンストラクトおよび少なくとも1つの環状ポリヌクレオチド修飾鋳型を、E.コリ(E.coli)細胞に提供する工程であって、前記E.コリ(E.coli)細胞が、誘導プロモータに機能可能なように結合したCas9エンドヌクレアーゼDNA配列を含み、前記Cas9エンドヌクレアーゼDNA配列が、前記E.コリ(E.coli)ゲノムのgalKゲノム配列内の標的部位に二重鎖切断を導入することができるCasエンドヌクレアーゼをコードし、前記環状ポリヌクレオチド修飾鋳型が、前記galKゲノム配列の少なくとも1つのヌクレオチド修飾を含む、工程;
b)(a)のE.コリ(E.coli)細胞から子孫細胞を増殖させる工程;および
c)前記少なくとも1つのヌクレオチド修飾の存在について(b)の子孫細胞を評価する工程を含む、方法。 - エケリキア・コリ(Escherichia coli:E.コリ(E.coli))細胞のゲノムの標的配列を編集するための方法であって、ガイドRNAをコードするDNA配列に機能可能なように結合したプロモータを含む少なくとも第1の組換えDNAコンストラクト、環状ポリヌクレオチド修飾鋳型、および誘導プロモータに機能可能なように結合したCas9エンドヌクレアーゼをコードするDNA配列を含む第2の組換えDNAコンストラクトをE.コリ(E.coli)細胞に提供する工程を含み、前記Cas9エンドヌクレアーゼが、前記E.コリ(E.coli)細胞のゲノムの標的配列に二重鎖切断を導入し、前記ポリヌクレオチド修飾鋳型が、前記標的配列の少なくとも1つのヌクレオチド修飾を含む、方法。
- 前記第1の組換えDNAコンストラクト、前記第2の組換えDNAコンストラクト、および前記環状ポリヌクレオチド修飾鋳型がそれぞれ、別個のプラスミドに提供される、請求項12に記載の方法。
- 前記第1の組換えDNAコンストラクト、前記第2の組換えDNAコンストラクト、および前記環状ポリヌクレオチド修飾鋳型が、1つのプラスミドに提供される、請求項12に記載の方法。
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JP6261500B2 (ja) | 2011-07-22 | 2018-01-17 | 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 | ヌクレアーゼ切断特異性の評価および改善 |
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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 |
US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
CA3075047C (en) | 2014-02-11 | 2022-02-01 | The Regents Of The University Of Colorado, A Body Corporate | Crispr enable method for multiplex genome editing |
WO2016022363A2 (en) | 2014-07-30 | 2016-02-11 | President And Fellows Of Harvard College | Cas9 proteins including ligand-dependent inteins |
US20190225955A1 (en) | 2015-10-23 | 2019-07-25 | President And Fellows Of Harvard College | Evolved cas9 proteins for gene editing |
CN114908093A (zh) * | 2016-02-26 | 2022-08-16 | 朗泽科技新西兰有限公司 | 用于c1固定菌的crispr/cas系统 |
LT3474669T (lt) | 2016-06-24 | 2022-06-10 | The Regents Of The University Of Colorado, A Body Corporate | Barkodu pažymėtų kombinatorinių bibliotekų generavimo būdai |
KR102547316B1 (ko) | 2016-08-03 | 2023-06-23 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | 아데노신 핵염기 편집제 및 그의 용도 |
AU2017308889B2 (en) | 2016-08-09 | 2023-11-09 | President And Fellows Of Harvard College | Programmable Cas9-recombinase fusion proteins and uses thereof |
EP3500671A4 (en) | 2016-08-17 | 2020-07-29 | The Broad Institute, Inc. | NEW CRISPR ENZYMS AND SYSTEMS |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
KR20240007715A (ko) | 2016-10-14 | 2024-01-16 | 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 | 핵염기 에디터의 aav 전달 |
BR112019012155A2 (pt) * | 2016-12-14 | 2019-11-12 | Stichting Technische Wetenschappen | uso de pelo menos uma molécula-guia de rna e uma proteína cas, método de ligação, clivagem, marcação ou modificação de um polinucleotídeo alvo de fita dupla, célula transformada, e, complexo de nucleoproteína |
US20190323019A1 (en) * | 2016-12-21 | 2019-10-24 | Yi-Hua Hsu | Composition for editing a nucleic acid sequence and method using the same |
US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | 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 |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
WO2018204777A2 (en) | 2017-05-05 | 2018-11-08 | The Broad Institute, Inc. | Methods for identification and modification of lncrna associated with target genotypes and phenotypes |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11453874B2 (en) | 2017-06-07 | 2022-09-27 | The Rockefeller University | Enhancement of CRISPR gene editing or target destruction by co-expression of heterologous DNA repair protein |
US9982279B1 (en) | 2017-06-23 | 2018-05-29 | Inscripta, Inc. | Nucleic acid-guided nucleases |
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CN109295054B (zh) * | 2017-07-25 | 2024-02-06 | 广州普世利华科技有限公司 | 用于靶向病原体基因RNA的gRNA及基于C2c2的病原体基因的检测方法及试剂盒 |
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) |
WO2019139645A2 (en) | 2017-08-30 | 2019-07-18 | President And Fellows Of Harvard College | High efficiency base editors comprising gam |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
CN109971778B (zh) * | 2017-12-27 | 2022-11-18 | 北京蓝晶微生物科技有限公司 | 一种在盐单胞菌中快速基因编辑的载体组合及其应用 |
DK3765615T3 (da) * | 2018-03-14 | 2023-08-21 | Arbor Biotechnologies Inc | Nye enzymer og systemer til målretning af crispr dna |
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 |
CA3109083A1 (en) * | 2018-08-09 | 2020-02-13 | G+Flas Life Sciences | Compositions and methods for genome engineering with cas12a proteins |
AU2019363487A1 (en) * | 2018-08-30 | 2021-04-15 | Inscripta, Inc. | Improved detection of nuclease edited sequences in automated modules and instruments |
CA3130488A1 (en) | 2019-03-19 | 2020-09-24 | David R. Liu | Methods and compositions for editing nucleotide sequences |
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GB2614813A (en) | 2020-05-08 | 2023-07-19 | Harvard College | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
WO2023125396A1 (en) * | 2021-12-27 | 2023-07-06 | Gracell Biotechnologies (Shanghai) Co., Ltd. | Systems and methods for cell modification |
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AU2001283377B2 (en) * | 2000-08-14 | 2007-09-13 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Enhanced homologous recombination mediated by lambda recombination proteins |
US7479385B2 (en) * | 2003-12-05 | 2009-01-20 | Wisconsin Alumni Research Foundation | Sugar kinases with expanded substrate specificity and their use |
WO2012164565A1 (en) * | 2011-06-01 | 2012-12-06 | Yeda Research And Development Co. Ltd. | Compositions and methods for downregulating prokaryotic genes |
PE20190844A1 (es) * | 2012-05-25 | 2019-06-17 | Emmanuelle Charpentier | Modulacion de transcripcion con arn de direccion a adn generico |
CN114634950A (zh) * | 2012-12-12 | 2022-06-17 | 布罗德研究所有限公司 | 用于序列操纵的crispr-cas组分系统、方法以及组合物 |
WO2015070193A1 (en) * | 2013-11-11 | 2015-05-14 | Liu Oliver | Compositions and methods for targeted gene disruption in prokaryotes |
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CN107250363A (zh) | 2017-10-13 |
EP3234117B1 (en) | 2021-03-03 |
BR112017012765A2 (pt) | 2018-01-16 |
KR102424626B1 (ko) | 2022-07-25 |
CN107250363B (zh) | 2021-03-30 |
AU2015363113B2 (en) | 2021-03-11 |
CA2971391A1 (en) | 2016-06-23 |
EP3234117A1 (en) | 2017-10-25 |
DK3234117T3 (da) | 2021-06-07 |
MX2017007907A (es) | 2017-09-18 |
US20170369866A1 (en) | 2017-12-28 |
KR20170087959A (ko) | 2017-07-31 |
WO2016099887A1 (en) | 2016-06-23 |
ES2865268T3 (es) | 2021-10-15 |
JP2017538422A (ja) | 2017-12-28 |
AU2015363113A1 (en) | 2017-06-29 |
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