JP2021524266A - 植物細胞におけるゲノム編集のためのv型crispr/ヌクレアーゼシステム - Google Patents
植物細胞におけるゲノム編集のためのv型crispr/ヌクレアーゼシステム Download PDFInfo
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- JP2021524266A JP2021524266A JP2021500272A JP2021500272A JP2021524266A JP 2021524266 A JP2021524266 A JP 2021524266A JP 2021500272 A JP2021500272 A JP 2021500272A JP 2021500272 A JP2021500272 A JP 2021500272A JP 2021524266 A JP2021524266 A JP 2021524266A
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Abstract
Description
本発明の方法、組成物、使用及び他の態様に関する各種用語は、本明細書及び特許請求の範囲の全体にわたって使用される。そのような用語には、特段の明示のない限り、本発明が関係する技術分野における通常の意味が付与されるものとする。他の詳細に定義された用語は、本明細書で提供された定義と一致する方法で解釈されるものとする。本明細書に記載のものと類似の又は同等の任意の方法及び材料を本発明の試験の実施において使用することができ、好ましい材料及び方法は本明細書に記載されている。
好ましくは、目的の遺伝子(GOI)は、特徴、好ましくは表現型の特徴を付与又は改変する遺伝子である。植物GOIは、したがって、好ましくは、植物の特徴、好ましくは表現型の植物の特徴を付与又は改変する。用語の「植物の特徴」とは、植物、植物細胞又は植物組織の任意の特徴を意味する。好ましくは、植物の特徴は、植物の発達、植物の成長、収量、バイオマス生産、植物の構造、植物生化学、植物生理学、代謝、生存能力、及びストレス耐性からなる群から選択される。或いは、又はさらに、植物の特徴は、DNA合成、DNA修飾、核内倍加、細胞期、細胞壁生合成、転写調節、シグナル伝達、貯蔵脂質動員、及び光合成からなる群から選択される。
本明細書で以下に詳述するのは、本明細書に記載された本発明の方法によって修飾することができる植物の特徴の非限定的な例である:
「成長」とは、バイオマスを拡大及び増加させる植物又は植物部分の能力を意味する。改変された成長とは、中でも、改変された成長速度、周期の時間、サイズ、植物の増殖又は増加を意味する。さらに及び/又は或いは、成長の特徴は、限定するものではないが、細胞周期(入口、進行、出口)、細胞分裂、細胞壁生合成及び/又はDNA合成、DNA修飾及び/又は核内倍加を含む、細胞プロセスを意味し得る。
MAD7−ヌクレアーゼ又はcrRNA−誘導型MAD7−ヌクレアーゼ複合体のタンパク質は、ユウバクテリウム・レクターレ(Eubacterium rectale)から得ることができるMAD7−ヌクレアーゼ、又はその任意のホモログ若しくはオルソログであり得る。好ましくは、MAD7−ヌクレアーゼは、配列番号1と少なくとも約50%、60%、70%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%又は100%の同一性を有するアミノ酸配列を有する。好ましくは、MAD7−ヌクレアーゼは、配列番号2と少なくとも約50%、60%、70%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%又は100%の同一性を有するヌクレオチド配列によってコードされているタンパク質である。好ましくは、MAD7−ヌクレアーゼは、配列番号74と少なくとも約50%、60%、70%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%又は100%の同一性を有するヌクレオチド配列によってコードされているタンパク質である。
一実施形態では、本明細書で定義したcrRNA−誘導型MAD7−ヌクレアーゼは、一本鎖切断又は二本鎖切断を導入することが可能である。この実施形態では、本発明の方法は、標的配列に少なくとも部分的に相補的である配列を有する一本鎖オリゴヌクレオチドを植物細胞に導入するステップをさらに含むことができる。このような一本鎖オリゴヌクレオチドはまた、テンプレートオリゴヌクレオチドとして注釈を付けることもできる。本発明の文脈では、「一本鎖」とは、その完全に相補的な鎖が存在しない、5’末端及び3’末端を有する、ヌクレオチドの直鎖ストレッチを意味する。
一実施形態では、本明細書で定義したcrRNA−誘導型MAD7−ヌクレアーゼは、一本鎖切断又は二本鎖切断を導入することができるが、例えば、その理由は、ヌクレアーゼが好ましくは内因性のヌクレアーゼ活性を含むためである。この実施形態では、本発明の方法は、本発明の方法によって修飾され、標的配列を含むDNAの配列に少なくとも部分的に相補的である配列を有する二本鎖オリゴヌクレオチド、二本鎖DNA、又は二本鎖DNA断片(ドナー断片)を植物細胞に導入するステップをさらに含むことができる。そのような二本鎖オリゴヌクレオチド又はDNAは、「ドナー構築物」又は「ドナーヌクレオチド」として本明細書で注釈が付けられ、これらは本明細書では互換的に使用される。
本発明の方法は、植物細胞のDNAをcrRNA−誘導型MAD7−ヌクレアーゼと接触させるステップを含む。これは、MAD7−ヌクレアーゼとMAD7−ヌクレアーゼをDNAに誘導するためのcrRNAとを植物細胞に導入することによって達成され得る。したがって、本発明の方法はまた、MAD7−ヌクレアーゼとMAD7−ヌクレアーゼをDNAに誘導するためのcrRNAとを植物細胞に導入するステップを含む、植物細胞のDNAを標的化修飾するための方法として定義することができる。MAD7−ヌクレアーゼ及びcrRNAは、MAD7−ヌクレアーゼ及び/若しくはcrRNA及び/若しくはcrRNA前駆体を細胞にコードするmRNAとして、又はMAD7−ヌクレアーゼタンパク質及び/若しくはcrRNAをコードする1つ又は複数の核酸として、MAD7−ヌクレアーゼタンパク質及び/若しくはcrRNA(好ましくは、事前に構築されたリボヌクレオ−タンパク質複合体として)の形態で、植物細胞に直接送達され得る。後者は、MAD7−ヌクレアーゼ及び/又はcrRNAをコードする遺伝子を含む1つ又は複数のベクターのトランスフェクションによって実施されてもよく、ベクターは前記遺伝子及び/又はcrRNAの一過性発現用である。植物細胞における導入又はトランスフェクションは、当技術分野で公知の任意の従来の方法により実施され得る。任意選択で、MAD7及び/又はcrRNA(複数可)をコードする配列は、植物細胞のゲノムに安定して導入される。MAD7−ヌクレアーゼ及びcrRNAがリボヌクレオ−タンパク質複合体として細胞内に送達される場合、本発明の方法は、前記複合体を植物細胞に導入するステップの前に、前記複合体を形成するステップをさらに含む。
本発明の方法は、本明細書で定義したMAD7−ヌクレアーゼ及びMAD7−ヌクレアーゼを誘導するためのcrRNAを植物細胞に導入するステップに先行して、前記植物細胞を提供するステップをさらに含んでいてもよい。当業者は、本発明の方法が特定の植物細胞タイプに限定されないことを理解する。特に、本明細書に開示した本発明の方法は、分裂細胞及び非分裂細胞に適用することができる。細胞は、トランスジェニックであっても非トランスジェニックであってもよい。植物細胞は、例えば、植物が再生され得る植物細胞組織培養物、植物カルス、植物塊、及び植物又は植物の一部、例えば、胚、花粉、胚珠、種子、葉、花、枝、果実、穀粒、穂、穂軸、殻、茎、根、根端、葯、粒などが無傷の植物細胞から得ることができる。
本方法は、標的化修飾を含む植物又はその子孫を再生するステップをさらに含み得る。好ましくは、そのような再生は、再生に適した条件を使用して実施される。当業者は、植物プロトプラストから植物を再生する方法及びプロトコルを十分に知っている。再生された植物の後代、子孫、バリアント、及び変異体もまた、これらの部分が本明細書で教示した方法で導入された標的化改変を含むならば、本発明の範囲内に含まれる。
本発明はまた、本発明の方法によって得ることができる植物細胞又は植物の後代に関する。さらに、本発明は、本明細書で定義した植物細胞又は植物から得ることができる植物産物、例えば、果実、葉、植物器官、植物油脂、植物油、植物デンプン、及び粉砕、磨砕されたか、完全なままであるか、他の材料と混合された、乾燥された、凍結された植物タンパク質画分に関する。これらの産物は繁殖しない物であり得る。好ましくは、前記植物産物は、
i)修飾されたDNA、好ましくは、本明細書で定義した修飾されたGOI、又はコードされたそれらの産物、
ii)本明細書で定義したMAD7−ヌクレアーゼ、及び
iii)MAD7−ヌクレアーゼをコードする配列
のうちの少なくとも1つ又は少なくとも一部を含む。
本発明はまた、キットオブパーツ、好ましくは、本明細書に記載した方法において使用するためのキットオブパーツに関する。好ましくは、キットオブパーツは、
本明細書で定義したMAD7−ヌクレアーゼを含む容器;
本明細書で定義したMAD7−ヌクレアーゼを誘導するためのcrRNA、本明細書で定義したODTNEのための一本鎖オリゴヌクレオチド、本明細書で定義したHRのための一本鎖オリゴヌクレオチド、又はそれらの任意の組合せを含む容器(好ましくは、容器は、異なる標的配列を標的とするための異なるガイド配列をそれぞれ含む2つ以上のcrRNAを含む);
本明細書で定義したMAD7−ヌクレアーゼをコードする1つ又は複数の核酸構築物又はベクターを含む容器(任意選択で、前記構築物又はベクターは、本明細書で定義した前記MAD7−ヌクレアーゼを誘導するための1つ又は複数のcrRNAをさらにコードする);及び、
本明細書で定義した前記MAD7−ヌクレアーゼを誘導するための1つ又は複数のcrRNAをコードする1つ又は複数の核酸構築物又はベクターを含む容器
のうちの少なくとも1つを含む。
トマトに標的化変異を生成するためのMAD7の使用
構築物
トマトでの翻訳に向けて最適化された、V型CRISPRヌクレアーゼMAD7及びAsCpf1の配列を、MAD7及びAsCpf1のタンパク質配列についてはそれぞれ配列番号3及び配列番号5により示し、Mad7及びAsCpf1タンパク質をコードするヌクレオチド配列についてはそれぞれ配列番号4及び配列番号6により示している。これらのORFを合成し、植物細胞で発現させるためにベクターで構成的35Sプロモーターの後ろにクローニングした。使用したcrRNAカセットの配列(U6プロモーター+crRNA)を表1に示す。これらもまた合成し、ベクターにクローニングした。すべてのプラスミド構築物を大腸菌(E.coli)に導入し、次いで形質転換したコロニーを使用して50mlの培養物に接種した。一晩増殖させた後、プラスミドDNAを、標準的な方法を使用してこれらの培養物から単離した。
トマト(Solanum lycopersicon)のMoneyberg種のin vitroシュート培養は、高プラスチック容器に0.8%寒天を含むMS20培地において、2000ルクスの光周期16/8hで、25℃及び60〜70%RHにて維持した。若葉(1g)を主脈(mid nerve)に対して垂直方向に静かにスライスし、酵素混合物の浸透を促進した。スライスした葉を酵素混合物(2%セルラーゼOnozuka RS、0.4%マセロザイムOnozuka R10を含むCPW9M)に移し、細胞壁消化を暗所で25℃にて一晩進行させた。プロトプラストを50μmのナイロン篩で濾過し、800rpmで5分間遠心分離することによって回収した。プロトプラストをCPW9M(Frearson、1973)培地に再懸濁し、3mLのCPW18S(Frearsonら 1973. Developmental Biology, 33:130〜137)を各チューブの底に長首ガラスパスツールピペットを使用して加えた。生存プロトプラストは、800rpmで10分間の遠心分離により、ショ糖とCPW9M培地の間の界面の細胞画分として回収した。プロトプラストをカウントし、MaMg(Negrutiuら 1987. Plant Molecular Biology, 8:363〜373)培地に1mLあたり106個の最終密度で再懸濁した。
全ゲノムDNAを、トマトプロトプラスト(トランスフェクションの48時間後)からDNeasy Plant Mini Kit(Qiagen)を使用して単離した。次いで、このgDNAをPCR反応で使用し、いずれかの標的領域を増幅した。次いで、PCR産物をテンプレートとして使用して各サンプルからライブラリーを生成し、次にこれをプールし、MiSeqプラットフォーム(Illumina)で126nt paired runを使用して配列決定した。各サンプルは、固有の5bpタグを使用して同定された。配列決定後、各サンプルから得た読み取りデータを処理して、標的部位に存在する配列変化の数及びタイプを同定した。標的部位に変異を有するカルスを同定するために、トランスフェクトされたプロトプラストを直径およそ3mmのカルスに再生させる。次いで、ダイレクトPCRキット(Phire Plant Direct PCRキット、Thermo Scientific)を遺伝子特異的プライマーと一緒に使用してそれぞれのカルスから標的配列を増幅する。次いで、得られたPCR産物を異なる方法で配列決定又は遺伝子型決定し、標的部位に変異を有するカルスを同定する。これらのカルスを次いで再生のために選択する。
本発明者らは、クラスV−CRISPRヌクレアーゼMAD7がゲノム植物細胞のINDEL変異を生成する能力を、トマトプロトプラストで異所的に7つの異なる配列を標的とするMAD7タンパク質及びcrRNAを発現させることにより試験した。最初に、トマトでのコドン利用に最適化されたMAD7 ORFを、C末端で融合した核局在化シグナル(NLS)とともに構築した。次いで、これを、植物細胞で発現させるために、構成的CaMV 35Sプロモーターの後ろにクローニングした。MAD7タンパク質は、標的配列に隣接するPAM配列、この場合にはTTTN、の存在を必要とし、これは、植物細胞並びに他の真核細胞のタイプで有効であることが報告されている別のクラスV−CRISPRヌクレアーゼ AsCpf1のPAMと同一である。この共通のPAM配列により、MAD7とAsCpf1の両方に全く同じ標的配列を含有するcrRNAを設計することができ、その結果、それぞれのヌクレアーゼによって生成される変異誘発効率の直接比較を実施することができる。MAD7又はAsCpf1のいずれかを発現するベクターを、トマト葉肉プロトプラストに、シロイヌナズナ(Arabidopsis thaliana)のU6プロモーターによって駆動されるcrRNAを発現する第2のベクターとともに導入した。そのようなシステムにおいて、MAD7又はAsCpf1 mRNAとcrRNAは、24〜48時間の短い期間で、高レベルで発現し、その時点で、導入されたプラスミドは細胞ヌクレアーゼによって分解され、CRISPR試薬は細胞から消失する。それらが存在している間は、それらはゲノムの特定の標的部位を見つけて、INDEL変異を生成することができる。導入されたプラスミドはゲノムに組み込まれることは稀であり、そのため、この手法はトランスジェニック系統をもたらさない。次いでプロトプラストを48時間培養した後、標的部位でのINDELの存在について配列決定することにより分析した。
Claims (15)
- DNAをcrRNA誘導型MAD7−ヌクレアーゼと接触させることを含む、植物細胞のDNAを標的化修飾するための方法。
- MAD7−ヌクレアーゼが2つの触媒的に活性なエンドヌクレアーゼドメインを含む、請求項1に記載の方法。
- MAD7−ヌクレアーゼが少なくとも1つの触媒的に不活性なエンドヌクレアーゼドメインを含む、請求項1に記載の方法。
- MAD7−ヌクレアーゼが機能性ドメイン、好ましくはデアミナーゼドメインに融合されている、請求項1〜3のいずれか一項に記載の方法。
- MAD7−ヌクレアーゼが、前記MAD7−ヌクレアーゼをコードする核酸構築物で細胞をトランスフェクトすることによって細胞に導入される、請求項1〜4のいずれか一項に記載の方法。
- MAD7−ヌクレアーゼが、MAD7−ヌクレアーゼで細胞をトランスフェクトすることによって細胞に導入される、請求項1〜4のいずれか一項に記載の方法。
- crRNAが、前記crRNAをコードする核酸構築物で細胞をトランスフェクトすることによって細胞に導入される、請求項1〜6のいずれか一項に記載の方法。
- crRNAが、crRNAで細胞をトランスフェクトすることによって細胞に導入され、好ましくはcrRNAが化学的に修飾されている、請求項1〜6のいずれか一項に記載の方法。
- 細胞がテンプレートオリゴヌクレオチドでさらにトランスフェクトされ、好ましくはテンプレートオリゴヌクレオチドが化学的に修飾されている、請求項1〜8のいずれか一項に記載の方法。
- 細胞がドナー構築物でさらにトランスフェクトされ、好ましくはドナー構築物が化学的に修飾されている、請求項1〜8のいずれか一項に記載の方法。
- MAD7−ヌクレアーゼ、crRNA、及び/又は任意選択でテンプレートオリゴヌクレオチド若しくはドナー構築物が、ポリエチレングリコール媒介性トランスフェクションを使用して、好ましくはPEGを含む水性媒体を使用して、植物細胞に導入される、請求項1〜10のいずれか一項に記載の方法。
- 標的化修飾を含む植物又はその子孫を再生するステップをさらに含む、請求項1〜11のいずれか一項に記載の方法。
- 好ましくはMAD7−ヌクレアーゼがcrRNAと複合体を形成している、植物細胞のDNAを標的化修飾するための、請求項1〜12のいずれか一項に定義したMAD7−エンドヌクレアーゼ、又はそれをコードする1つ若しくは複数の構築物。
- i)請求項13に記載のMAD7−エンドヌクレアーゼを含む容器;及び
ii)請求項13に記載の1つ又は複数の構築物を含む容器、
のうちの少なくとも1つ、並びに任意選択で、1つ若しくは複数のcrRNA及び/又はそれをコードする構築物を含む容器
を含む、植物細胞のDNAを標的化修飾するためのキット。 - 植物細胞のDNAを標的化修飾するための、請求項13で定義したcrRNA誘導型MAD7−エンドヌクレアーゼ、又はそれをコードする1つ若しくは複数の構築物、又は請求項14で定義したキットの使用。
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WO2021257716A2 (en) * | 2020-06-16 | 2021-12-23 | Bio-Techne Corporation | Engineered mad7 directed endonuclease |
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WO2022236147A1 (en) * | 2021-05-06 | 2022-11-10 | Artisan Development Labs, Inc. | Modified nucleases |
WO2023169093A1 (zh) * | 2022-03-10 | 2023-09-14 | 青岛清原化合物有限公司 | 工程化核酸酶及其应用 |
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WO2020011985A1 (en) | 2020-01-16 |
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