JP2016512695A - オリゴヌクレオチド仲介型遺伝子修復を使用した標的遺伝子修飾の効率を高めるための方法および組成物 - Google Patents
オリゴヌクレオチド仲介型遺伝子修復を使用した標的遺伝子修飾の効率を高めるための方法および組成物 Download PDFInfo
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Abstract
Description
1つまたは複数の無塩基部位ヌクレオチド、
1つまたは複数の8’オキソdAおよび/または8’オキソdGヌクレオチド、
その3’末端における逆向き塩基、
1つまたは複数の2’O−メチルヌクレオチド、
その5’末端における1つまたは複数、および好ましくは2、3、4、5、6、7、8、9、10個、またはそれより多くの2’O−メチルRNAヌクレオチド、
挿入色素、
5’末端キャップ、
ホスホチオエート修飾、メチルホスホネート修飾、ロックド核酸(LNA)修飾、O−(2−メトキシエチル)(MOE)修飾、ジPS修飾、およびペプチド核酸(PNA)修飾からなる群から選択される骨格修飾、
1つまたは複数の鎖間架橋、
好ましくはGRONの5’または3’末端に1つまたは複数の共有結合した蛍光色素、
ハイブリダイゼーションエネルギーを増大させる1つまたは複数の塩基を含み得る。この一覧は限定的であることを意味しない。
本発明の理解を容易にするため、幾つかの用語を以下のように定義する。
本明細書で開示している核酸分子(例えば、部位特異的ヌクレアーゼ、またはCRISPRのガイドRNA)は、組換え核酸構築物の生成において使用することができる。一実施形態では、本開示の核酸分子を、核酸構築物、例えば対象植物における発現用の発現カセットの調製において使用することができる。例えば構築物が宿主ゲノム中に組み込まれない、またはそれが組み込まれた状態の場合はプロモーターによってもたらされる制御下で宿主のゲノム内の構築物の位置に維持されるとき、この発現は一過的である可能性がある。
本発明は一般に、ゲノムまたは他のヌクレオチド配列中の特定位置に対する、修飾の標的化の効率を改善するための新規な方法に関する。さらに本発明は、本明細書で開示している手法により修飾、突然変異または顕在化された標的DNAに関する。本発明はさらに、本発明の方法によって修飾された細胞、組織、および生物に関する。本発明は、成功した変換システム、the Rapid Trait Development System(RTDS(商標)、Cibus US LLC)と部分的に関連する組成物および方法の開発に基づく。
本発明は、修復オリゴヌクレオチドを使用する、標的遺伝子の転換の有効性を高めるための幾つかの手法を記載し、それらは単独でまたは互いに組み合せて使用することができる。これらは以下を含む:
1.標的(ミスマッチ)部位にDNA修復機構を向ける修復オリゴヌクレオチドに対する修飾の導入。
A.オリゴヌクレオチド(例えば、10塩基以内、およびより好ましくは所望ミスマッチ部位の5塩基)における1つまたは複数の無塩基部位の導入により塩基切除修復(BER)中の中間体である損傷が生じ、それによってBER機構を修復オリゴヌクレオチドによる転換標的の部位近辺に向ける。dスペーサー(無塩基フラン)修飾オリゴヌクレオチドは、例えばTakeshita et al.,J.Biol.Chem.,262:10171−79,1987中に記載されたように調製することができる。
B.オリゴヌクレオチド中へのまたはオリゴヌクレオチドと一緒のいずれかの、一本鎖または二本鎖切断を誘導する化合物の封入によって、非相同末端結合(NHEJ)、マイクロホモロジー仲介末端結合(MMEJ)、および相同組換えにより修復される損傷が生じる。例えば、ブレオマイシンファミリーの抗生物質、ジンクフィンガー、FokI(または任意のIIS型クラスの制限酵素)および他のヌクレアーゼを修復オリゴヌクレオチドの3’末端または5’末端に共有結合させて、修復オリゴヌクレオチドによる転換標的の部位近辺に二本鎖切断を導入することが可能である。ブレオマイシンファミリーの抗生物質は、ブレオマイシン、ゼオシン、フレオマイシン、タリソマイシン、ペプレオマイシンおよびその他を含めたDNA切断糖ペプチドである。
C.オリゴヌクレオチド(例えば、10塩基以内、およびより好ましくは所望ミスマッチ部位の5塩基)に取り込まれる1つまたは複数の8’オキソdAまたはdGの導入によって、反応性酸素種によって生成する損傷と類似した損傷が生じる。これらの損傷はいわゆる「助長型修復」系を誘導する。例えば、Kim et al.,J.Biochem.Mol.Biol.37:657−62,2004を参照。
2.修復オリゴヌクレオチドの安定性の増大:
修復オリゴヌクレオチド上に3’ブロッキング末端を作製するためのオリゴヌクレオチドの3’末端における逆向き塩基(idC)の導入。
修復オリゴヌクレオチドの5’および/または3’末端における、ハイブリダイゼーションエネルギーを増大させる1つまたは複数の2’O−メチルヌクレオチドまたは塩基の導入(例えば、WO2007/073149参照)。
修復オリゴヌクレオチドの5’末端における複数の2’O−メチルRNAヌクレオチドの導入、所望のミスマッチ部位をもたらすDNA塩基が生じ、それによって岡崎フラグメント様核酸構造を生成する。
アクリジン、ソラレン、臭化エチジウムおよびサイバー染色液などの結合(5’または3’)挿入色素。
T/Aクランプ、コレステロール成分、SIMA(HEX)、リボCおよびアミダイトなどの5’末端キャップの導入。
ホスホチオエート、2’O−メチル、メチルホスホネート、ロックド核酸(LNA)、(MOE)(メトキシエチル)、ジPSおよびペプチド核酸(PNA)などの骨格修飾。
例えばシスプラチンおよびマイトマイシンCなどの鎖間架橋試薬物質による、修復オリゴヌクレオチドの架橋。
Cy3、DY547、Cy3.5、Cy3B、Cy5およびDY647などの蛍光色素との結合。
3.ハイブリダイゼーションエネルギーを増大させる塩基の取り込みによる、修復オリゴヌクレオチドのハイブリダイゼーションエネルギーの増大(例えば、WO2007/073149参照)。
4.合成用の構成単位としてヌクレオチドマルチマー(ジマー、トリマー、テトラマーなど)を使用することによる、修復オリゴヌクレオチド合成の質の向上。これによって、より少ないカップリングステップ、および構成単位からの完全長産物の容易な分離をもたらす。
5.好ましくは修復オリゴヌクレオチド中に2個以上の標的突然変異がある、長鎖修復オリゴヌクレオチド(すなわち、55ヌクレオチド長を超える、好ましくは75〜300ヌクレオチド長、より好ましくは少なくとも100ヌクレオチド長、さらにより好ましくは少なくとも150ヌクレオチド長、および最も好ましくは少なくとも200ヌクレオチド長)の使用。
植物細胞を形質転換するのに使用される任意の一般に知られる方法を、遺伝子修復オリゴ核酸塩基の送達に使用することができる。例示的な方法を以下に列挙する。本発明は、1つまたは複数のDNA修飾試薬で細胞をトランスフェクトするための多くの方法を企図する。実際本発明は、任意の特定の方法に限られない。1つまたは複数の細胞にDNA修飾試薬を導入するための方法は当技術分野でよく知られており、マイクロインジェクション、エレクトロポレーション、受動吸着、リン酸カルシウム−DNA共沈殿法、DEAEデキストラン仲介トランスフェクション、ポリブレン仲介トランスフェクション、リポソーム融合、リポフェクション、ヌクレオフェクション、プロトプラスト融合、レトロウイルス感染、バイオリスティクス(すなわち、パーティクルボンバードメント)などだけには限られないが、これらを含む。
様々な実施形態において、本明細書で開示している植物は、高木もしくは低木として成長する任意の樹木植物種、任意の草本植物種、または可食果実、種子もしくは野菜を生成する任意の種、または有色もしくは芳香性品種の花を生成する任意の種を含めた、任意の種の双子葉植物、単子葉植物または裸子植物であってよい。例えば植物は、それらが既に具体的に言及されていない限り、キャノーラ、ヒマワリ、コーン、タバコ、テンサイ、ワタ、メイズ、コムギ、オオムギ、イネ、アルファルファ、オオムギ、モロコシ、トマト、マンゴー、モモ、リンゴ、ナシ、イチゴ、バナナ、メロン、ジャガイモ、ニンジン、レタス、タマネギ、ダイズ、ダイズ種、サトウキビ、マメ科植物、ヒヨコマメ、フィールドピー、マメ科マメ、レンズマメ、カブ、スウェーデンカブ、芽キャベツ、ルピナス、カリフラワー、ケール、エンドウマメ、ポプラ、マツ、ユーカリノキ、ブドウ、カンキツ属、ライコムギ、アルファルファ、ライムギ、オートムギ、芝生と飼草、アマ、ナタネ、カラシナ、キュウリ、アサガオ、バルサム、コショウ、ナス、マリゴールド、ロータス属、キャベツ、デージー、カーネーション、チューリップ、アヤメ属、ユリ、および堅果産生植物からなる群の植物種から選択することができる。
植物種の様々な組織の組織培養、およびそこからの植物の再生は知られている。例えば、組織培養によるキャノーラ品種の繁殖は、以下のいずれか、Chuong et al.,「A Simple Culture Methods for Brassica hypocotyls Protoplasts,」Plant Cell Reports4:4−6,1985;Barsby,T.L.et al.,「A Rapid and Efficient Alternative Procedure for the Regeneration of Plants from Hypocotyl Protoplasts of Brassica napus,」Plant Cell Reports(Spring,1996);Kartha,K.,et al.,「In vitro Plant Formation from Stem Explants of Rape,「Physiol.Plant,31:217−220,1974;Narasimhulu,S.,et al.,「Species Specific Shoot Regeneration Response of Cotyledonary Explants of Brassicas,」Plant Cell Reports(Spring1988);Swanson,E.,「Microspore Culture in Brassica,」Methods in Molecular Biology,Vol.6,Chapter17,p.159,1990だけには限られないが、これらのいずれかに記載されている。
Sommer et al.,(Mol Biotechnol.33:115−22,2006)は、一ヌクレオチド変化を利用し緑色蛍光タンパク質(GFP)変異体において青色蛍光と緑色蛍光を転換する、in vivo遺伝子転換を検出するためのレポーターシステムを記載する。このレポーターシステムを、モデル種としてシロイヌナズナ(Arabidopsis thaliana)を使用しGRON長修飾後のGRON転換の効率を評価した、以下の実験中での使用に適合させた。
この実験系の目的は、(GRONの各末端に3PS成分を有する)ホスホチオエート(PS)標識GRONと、5’Cy3/3’idC標識GRONの効率を比較することである。5’Cy3/3’idC標識GRONは、5’Cy3フルオロフォア(アミダイト)および3’idC逆向き塩基を有する。青色蛍光タンパク質(BFP)から緑色蛍光への転換を使用して効率を評価した。
この実験系の目的は、DNA切断を誘導するブレオマイシンファミリーのメンバー、ゼオシン(商標)(1mg/ml)の有無の下で、GRONの各末端に3PS成分を有するホスホチオエート(PS)標識GRONと「岡崎フラグメントGRON」の転換効率を比較することである。これらのGRONの設計は図2中に示す。GRONはPEG処置によりシロイヌナズナBEPプロトプラストに送達し、BFPからGFPへの転換は処置後24時間でサイトメトリーにより測定した。ゼオシン(1mg/ml)で処置したサンプルは、PEG処置前に氷上で90分間ゼオシンとインキュベートした。
この実験系の目的は、(ゼオシンの有無の下で)、異なる長さのGRONの各末端に3PS成分を有するホスホチオエート(PS)標識GRONの転換効率を比較することであった。41量体、101量体、および201量体を表1中に示す。再度、サイトメトリーにより測定して、ゼオシン(1mg/ml)の存在によってBFPからGFPへの転換率は増大した(表3)。3つの実験全てにおける大まかな傾向は、ゼオシンの存在下と不在下の両方でNCGRON長の増大に比例した。ゼオシン存在下でのBFP−4/NC/101とBFP−4/C/101以外、これは41量体NCGRONとほぼ等しいがそれよりは低い転換率を有していた。これは、BFP−4/41コードおよび非コードGRONを使用した前の全ての実験とは対照的であり、この場合非コードGRONは常にコードGRONより優れていた。転換頻度のこの非対称性は、この実験系で使用したBFP−4/201GRONにも当てはまった。
CRISPR複合体を構築する際に、3つの設計成分、Cas9、gRNA(ガイドRNA)、および標的領域(内在標的遺伝子中のプロト−スペーサー)を考慮に入れなければならない。
− それぞれ35Sまたはコーンユビキチンによって誘導される、シロイヌナズナまたはコーンに最適化させた化膿性連鎖球菌(Streptococcus pyogenes)コドンからのCas9遺伝子の一過的発現。最適化遺伝子はGenewizまたはDNA2.0によって合成された。NBは隠れイントロンが生成されないことを確実にしなければならない。
− G1155に従うRBCSE9ターミネーター
− C末端融合体としての1つのSV40NLS(PKKRKV)
− ベクター骨格は、いずれも本発明者らの一過的発現系−G1155に従うものである。
− LeCong et al.,2013およびJinek et al.,2013に従うキメラトレーサーRNA−プレ−creRNAの使用の提案。LeCong et alが、原型完全長トレーサー+プレ−creRNA複合体が、キメラ型よりはるかに効率良く切断したことを示したことを記す。したがって一つの選択肢は、完全長(89bp)トレーサーRNAを使用したキメラの作製である。
− gRNAの配列((N)20はガイド配列を表す)。括弧付きの配列は完全長89bp型を含む。
NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG(TTATGTTCTTGAAAAAAGTGAGTGGCACCGAGTCGGTGGTGCTTTTTT)
− G1155に従うRBCSE9ターミネーターまたはWang et al.2013に従う一連のターミネーター、および一成分手法を以下に示す。
標的領域
−ガイド配列の特異性を標的領域配列によって定義する。モデル生物の選択とは無関係に、これはBFPのY66H遺伝子座である。Y66H近辺のPAM(NGG)配列は唯一の設計制限配列である。さらに、3’における12bpのガイド配列にY66H位置(「シード配列」)を含めることは、修復が実施された後、その部位は再度切断されないことを意味する。
LeCong et al.(2013)は、以下に略述するような、polIIIU6プロモーターの誘導により1つの一過的構築物としてgRNAとCas9の両方を発現させる、簡略化された手法を使用した。このように、所与の穀物に関して、ガイド挿入配列を単に交換することによって多数の遺伝子を標的化することができる。本発明者らは、EF1αプロモーターを穀物に適したプロモーターと交換する(pMASとAt、UbiとZim)。ターミネーターに関して、本発明者らはRBCSE9を使用する。植物において使用するNLSは、前に略述した1つのC末端SV40である。
一過的選択肢
− 植物において標的認識およびヌクレアーゼ活性を確認するための一手法は、TALENに関してZhang et al.(2013)が使用したYFP一本鎖アニーリングアッセイに匹敵する。スペーサー配列(標的配列)およびPAMはYFPまたは同等遺伝子に挿入する必要がある。
一過的選択肢
− TALEN−BFPシステムを対照として使用することが可能である。
− 前述の手法は、所与のスペーサー配列に関する所与のCRISPRシステムの機能を確認するための現在のツールであるが、植物におけるCRISPRの活性の概念を証明するのはGFPシステムの使用である。
− 本明細書においてBFP→GFPに使用した設計は、G1155と一緒に、およびGRONなしでAtに同時形質転換することが可能である。切断が十分有効であった場合、GFP発現の低下は明らかであると思われる。これはおそらく、プラスミドローディングの最適化を必要とする可能性が高い。
− 活性を確認した後、ゲノムBFP標的を映像および配列ベースの読み出しで標的化する。
− CRISPRシステムの活性を迅速に確認するため、Jinek et al.2012に従いin vitroアッセイを使用することが可能である。本明細書において予め作製し精製した化膿性連鎖球菌Cas9を、合成gRNAおよび認識配列含有プラスミドと共にインキュベートする。切断の成功はゲル電気泳動により分析し切断プラスミドを検索する。
詳細なプロトコール:
CRISPR認識配列の柔軟性を考慮すると、3’NGGPAM配列によって定義される潜在的プロトスペーサー配列を見つけるのは難しくない。
ZmEPSPS
認識される。したがって、好ましい実施形態および任意選択の特徴によって本発明を具体的に開示してきたが、本明細書で開示している概念の修正および変更は当業者に委ねることができ、このような修正および変更は、添付の特許請求の範囲によって定義する本発明の範囲内にあると考えられることが理解されるはずである。
Claims (10)
- 細胞中の標的デオキシリボ核酸(DNA)配列に遺伝子修復オリゴ核酸塩基(GRON)仲介型突然変異を導入するための方法であって、細胞へのGRONの送達を含み、GRONが以下の特徴:
GRONが55塩基長より大きく、GRONが標的DNAに導入するための2つ以上の突然変異部位を場合によっては含むこと、
GRONが1つまたは複数の無塩基部位ヌクレオチドを含むこと、
GRONが1つまたは複数の8’オキソdAおよび/または8’オキソdGヌクレオチドを含むこと、
GRONがその3’末端に逆向き塩基を含むこと、
GRONがその5’または3’末端に1つまたは複数の2’O−メチルヌクレオチドを含むこと、
GRONがその5’末端に1つまたは複数の2’O−メチルRNAヌクレオチドを含むこと、
GRONがその5’末端に少なくとも2つの2’O−メチルRNAヌクレオチドを含むこと、
GRONが挿入色素を含むこと、
GRONが5’末端キャップを含むこと、
GRONがホスホチオエート修飾、メチルホスホネート修飾、ロックド核酸(LNA)修飾、O−(2−メトキシエチル)(MOE)修飾、ジPS修飾、およびペプチド核酸(PNA)修飾からなる群から選択される骨格修飾を含むこと、
GRONが1つまたは複数の鎖間架橋を含むこと、
GRONが1つまたは複数の共有結合した蛍光色素を含むこと、および
GRONがハイブリダイゼーションエネルギーを増大させる1つまたは複数の塩基を含むこと、
の1つまたは複数、および好ましくは2、3、4、5つ、またはそれより多くを含む方法。 - ヌクレオチド多量体を使用してGRONの全体または一部分を合成するステップをさらに含む、請求項1に記載の方法。
- 標的デオキシリボ核酸(DNA)配列が植物細胞ゲノム内に存在する、請求項1または2のいずれか一項に記載の方法。
- 植物細胞が、キャノーラ、ヒマワリ、コーン、タバコ、テンサイ、ワタ、メイズ、コムギ、オオムギ、イネ、アルファルファ、オオムギ、モロコシ、トマト、マンゴー、モモ、リンゴ、ナシ、イチゴ、バナナ、メロン、ジャガイモ、ニンジン、レタス、タマネギ、ダイズ、ダイズ種、サトウキビ、マメ科植物、ヒヨコマメ、フィールドピー、マメ科マメ、レンズマメ、カブ、スウェーデンカブ、芽キャベツ、ルピナス、カリフラワー、ケール、エンドウマメ、ポプラ、マツ、ユーカリノキ、ブドウ、カンキツ属、ライコムギ、アルファルファ、ライムギ、オートムギ、芝生と飼草、アマ、ナタネ、カラシナ、キュウリ、アサガオ、バルサム、コショウ、ナス、マリゴールド、ロータス属、キャベツ、デージー、カーネーション、チューリップ、アヤメ属、およびユリからなる群から選択される種である、請求項1から3のいずれか一項に記載の方法。
- 植物細胞がトランスジェニックである、請求項1から4のいずれか一項に記載の方法。
- 標的DNA配列が植物細胞の内在性遺伝子である、請求項1から5のいずれか一項に記載の方法。
- 植物細胞からGRONによって導入された突然変異を有する植物を再生するステップをさらに含む、請求項1から6のいずれか一項に記載の方法。
- 植物から種子を回収するステップをさらに含む、請求項7に記載の方法。
- 請求項1から6のいずれか一項に記載の方法に従いGRONによって導入されたゲノム修飾を含む植物細胞、または請求項7に記載の方法に従いGRONによって導入されたゲノム修飾を含む植物、または請求項8に記載の方法に従いGRONによって導入されたゲノム修飾を含む種子。
- 細胞が、植物細胞、動物細胞、細菌細胞、酵母細胞および真菌細胞からなる群から選択される1つまたは複数の細胞である、請求項1に記載の方法。
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