JP6704251B2 - Fad2性能座および標的化切断を誘導することができる対応する標的部位特異的結合タンパク質 - Google Patents
Fad2性能座および標的化切断を誘導することができる対応する標的部位特異的結合タンパク質 Download PDFInfo
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Description
本出願は、これによりその開示の全体が参照により組み込まれる、2012年9月7日出願の米国仮特許出願第61/697,886号の利益に対する優先権を主張するものである。
[1]部位特異的様式で大豆細胞中のFAD2遺伝子中の標的部位を切断して、それによって上記FAD2遺伝子中の破壊を引き起こす工程を含む、大豆細胞のFAD2遺伝子を修飾する方法であって、上記FAD2遺伝子が切断後に修飾される方法。
[2]対象となる核酸配列を上記破壊に組み込む工程をさらに含む、上記[1]に記載の方法。
[3]上記FAD2遺伝子がFAD2 2.3、FAD2 2.6遺伝子、または両方である、上記[1]または上記[2]に記載の方法。
[4]部位特異的様式での上記切断が、DNA結合ドメイン、および切断ドメインまたは切断ハーフドメインを含む融合タンパク質或いは上記融合タンパク質をコードするポリヌクレオチドを上記細胞に導入する工程を含み、上記融合タンパク質が特異的に上記標的部位に結合し、上記標的部位またはその近くを切断して、それによって上記破壊を引き起こす、上記[1]から[3]のいずれかに記載の方法。
[5]上記DNA結合ドメインが、メガヌクレアーゼDNA結合ドメイン、ロイシンジッパーDNA結合ドメイン、転写活性化物質様(TAL)DNA結合ドメイン、RNAガイド化CRISPR−Cas9、リコンビナーゼ、ジンクフィンガータンパク質DNA結合ドメイン、および上記のいずれかのキメラ組み合わせからなる群から選択される、上記[4]に記載の方法。
[6]上記切断ドメインまたは切断ハーフドメインがIIS型制限エンドヌクレアーゼからの切断ハーフドメイン、FokIエンドヌクレアーゼからの切断ハーフドメイン、StsIエンドヌクレアーゼからの切断ハーフドメイン、およびホーミングエンドヌクレアーゼからなる群から選択される、上記[4]または上記[5]に記載の方法。
[7]上記融合タンパク質がジンクフィンガーヌクレアーゼである、上記[4]から[6]のいずれかに記載の方法。
[8]上記ジンクフィンガーヌクレアーゼが3〜6個のジンクフィンガードメインを含み、各ジンクフィンガードメインが認識ヘリックス領域を含み、上記ジンクフィンガータンパク質が表2の一列に整列され、示される上記認識ヘリックス領域を含む、上記[7]に記載の方法。
[9]部位特異的様式での上記切断がFAD2 2.3およびFAD2 2.6のいくつかであるが全てではないコピーに特異的である、上記[1]から[8]のいずれかに記載の方法。
[10]上記標的部位が配列番号14〜配列番号20からなる群から選択される、上記[1]から[9]のいずれかに記載の方法。
[11]対象となる上記核酸配列が標的部位に結合するDNA結合ドメインを含む配列、1つまたはそれ以上の殺虫剤耐性遺伝子、1つまたはそれ以上の除草剤耐性遺伝子、1つまたはそれ以上の窒素利用効率遺伝子、1つまたはそれ以上の水利用効率遺伝子、1つまたはそれ以上の栄養価遺伝子、1つまたはそれ以上のDNA結合遺伝子、1つまたはそれ以上の選択可能なマーカー遺伝子、およびこれらの組み合わせからなる群から選択される、上記[2]から[10]のいずれかに記載の方法。
[12]上記[1]から[11]のいずれかに記載の方法によって修飾された大豆細胞を含む大豆細胞または大豆植物。
[13]大豆細胞または植物が、FAD2 2.3および/またはFAD2 2.6遺伝子の1つまたはそれ以上のコピーに組み込まれた対象となるヌクレオチド配列を含むトランスジェニック細胞または植物である、上記[12]に記載の細胞または植物。
[14]上記ヌクレオチド配列が上記細胞と異種性または相同性である、上記[13]に記載の細胞または植物。
[15]上記相同性配列が少なくとも1つの一塩基多型を含む、上記[14]に記載の細胞または植物。
[16]上記核酸配列が配列番号14〜配列番号20からなる群から選択される標的部位またはその近くで組み込まれる、上記[13]から[15]のいずれかに記載の細胞または植物。
[17]配列番号14〜配列番号20からなる群から選択される核酸標的部位またはその近くを切断する、部位特異的ジンクフィンガーヌクレアーゼ。
[18]上記ジンクフィンガーヌクレアーゼが3〜6個のジンクフィンガードメインを含み、各ジンクフィンガードメインが認識ヘリックス領域を含み、上記ジンクフィンガータンパク質が表2の一列に整列され、示される上記認識ヘリックス領域を含む、上記[17]に記載のジンクフィンガーヌクレアーゼ。
本開示は、(例えば、植物、藻類および真菌類において)FAD2遺伝子の発現を調節するための組成物および方法、ならびに対象となる核酸配列(例えば、外因性核酸配列)の宿主細胞への標的化組込みのための部位としてのこれらの座の使用を記載する。いくつかの実施形態では、宿主細胞が、そのいずれかまたは全てが選択的に修飾および/または破壊され得る1つまたはそれ以上のFAD2配列(例えば、ホモログまたはパラログ)を含む1つまたはそれ以上のゲノムを含み得る。具体的な例では、本開示が、ダイズ(Glycine max)(例えば、ダイズ栽培品種Jack、Williams 82、X5、WestagおよびMaverick)のFAD2 2.3およびFAD2 2.6遺伝子、ならびに対応するホモログまたはパラログ、ならびに対象となる核酸配列の標的化組込みのための座としてのその使用を記載する。本明細書に記載されるように、FAD2遺伝子は宿主の脂肪酸生合成に関与しているが、(例えば、FAD2コード配列への外因性核酸の組込みによる)修飾または破壊は、結果として生じる宿主生物に予想外に全く有害効果をもたらさないまたは最小の有害効果しかもたらし得ない。
核酸配列は、米国特許法施行規則第1.822条に定義されるヌクレオチド塩基についての標準的な文字略語を用いて示される。1本鎖のみの各核酸配列が示されるが、示される鎖への任意の言及により、相補鎖が含まれると理解される。
本発明の実施形態は、組み込まれた核酸によって影響を及ぼされるものを超えて宿主の他の表現型に大いに悪影響を及ぼすことのない、外因性核酸(例えば、導入遺伝子)の宿主ゲノムへの標的化組込みのための手法を確立する。単一の宿主ゲノム中の複数の核酸を「スタッキングする」ために、いくつかの実施形態が使用され得る。このような手法は、4つの相互接続技術の開発および配置を要する:特異的ゲノムDNA位置への二本鎖切断の導入を可能にする標的化技術(例えば、Puchta et al.(1993) Nucleic Acids Res.21:5034-40;Siebert and Puchta (2002) Plant Cell 14:1121-31;D'Halluin et al.(2008) Plant Biotechnol.J.6(1):93-102;Cai et al.(2009) Plant Mol.Biol.69(6):699-709;Shukla et al.(2009) Nature 459(7245):437-41);Shan et al.(2103) Nature Biotechnol.31:686-680;Le et al.(2013) Nature Biotechnol 31: 688-691;Nekrasov et al.(2013) Nature Biotechnol.31:691-693、Ainely et al.(2013) Plant Biotechnol.J.(On Line 19 Aug)参照);最適化外因性(ドナー)核酸の送達を可能にする送達技術(Bibikova et al.(2003) Science 300(5620):764);標的化ドナーDNA挿入のためにHDRまたはNHEJ頻度を増加させるための、(相同組換えまたはNHEJ経路のいずれかに位置する)宿主遺伝子の修飾を伴う組込み技術;標的化組込みイベントを富化し特徴付けるための分析ツール;および遺伝的に十分定義されかつ形質転換された宿主生物に大いに悪影響を及ぼすことなく世代にわたる安定な遺伝子発現を支持する特異的な所望の宿主ゲノム位置(「性能座」)。また、米国特許出願公開第20030232410号明細書;第20050208489号明細書;第20050026157号明細書;第20050064474号明細書;第20060188987号明細書;第20090263900号明細書;第20090117617号明細書;第20100047805号明細書;第20110207221号明細書;第20110301073号明細書;第2011089775号明細書;第20110239315号明細書;第20110145940号明細書;第20080182332号明細書;第20090205083号明細書;第20100199389号明細書;第20110167521号明細書も参照されたい。例えば、植物では、性能座は、座で導入遺伝子が挿入されたトランスジェニック植物の農学的または品質特性に対する負の影響が無視できるまたは存在しない座である。
特許請求の範囲を含む本出願で使用される場合、例えば、「a」、「an」および「the」という単数および単数形の用語は、別段の明確な指示がない限り、複数指示対象を含む。したがって、例えば、「植物(plant)」、「植物(the plant)」または「植物(a plant)」への言及は、複数の植物も指す。さらに、文脈に応じて、「植物(plant)」という用語の使用は、その植物の遺伝的に類似または同一の子孫も指し得る。同様に、「核酸」という用語は、核酸分子の多くのコピーを指し得る。同様に、「プローブ」という用語は、多くの類似または同一のプローブ分子を指し得る。
FAD2(脂肪酸デサチュラーゼ2)と命名される座は、植物中の脂肪酸含量の複雑な多遺伝子形質の遺伝に関与するQTLに含まれる。FAD2は、オレイン酸(18:1)のリノール酸(C18:2)への不飽和化を担う酵素をコードしている。Tanhuanpaa et al.(1998) Mol. Breed. 4:543-50; Schierholt et al.(2001) Crop Sci. 41:1444-9。
C18:0 → C18:1 → C18:2 → C18:3
FAD2 FAD3
FAD2遺伝子は、それだけに限らないが、トウモロコシ、大豆、ワタ、シロイヌナズナ、コムギ、イネ科牧草、イネ、ヒマワリおよびアブラナ属を含む主要な植物および藻類の種で同定されており、FAD2発現の修飾は、このような生物における脂肪酸プロファイルの変化をもたらす。さらに、修飾FAD2遺伝子を含む植物が商品化されており、FAD2遺伝子の破壊は、宿主植物への農学的不利益なしに宿主植物によって産生される油の栄養的および機能的特性を改善することができることが示されている。例えば、Nexera(登録商標)ブランド(Dow AgroSciences,LLC)で商品化されているアブラナおよびヒマワリ品種は、野生型アブラナおよびヒマワリのプロファイルと比べると、高いオレイン酸、低いリノール酸および低いリノレン酸(および低い飽和脂肪酸)組成によって特徴付けられる。
FAD2座における外因性核酸の部位特異的組込みは、当業者に公知の任意の技術によって達成され得る。いくつかの実施形態では、FAD2座における外因性核酸の組込みが、細胞(例えば、単離細胞または組織もしくは生物中の細胞)を外因性核酸を含む核酸分子と接触させることを含む。例では、このような核酸分子が、核酸分子と少なくとも1つのFAD2座との間の相同組換えを促進する外因性核酸に隣接するヌクレオチド配列を含み得る。特定の例では、相同組換えを促進する外因性核酸に隣接するヌクレオチド配列が、FAD2座の外因性ヌクレオチドと相補的であり得る。特定の例では、相同組換えを促進する外因性核酸に隣接するヌクレオチド配列が、予め組み込まれた外因性ヌクレオチドと相補的であり得る。いくつかの実施形態では、複数の外因性核酸が、1つのFAD2座において、例えば、遺伝子スタッキングで組み込まれ得る。
いくつかの実施形態では、部位特異的組込みが、例えば、宿主生物のゲノム中の特定のヌクレオチド配列を認識し、これに結合することができる因子を利用することによって達成され得る。例えば、多くのタンパク質が、部位特異的様式でDNAを認識し、これに結合することができるポリペプチドドメインを含む。DNA結合ポリペプチドによって認識されるDNA配列は、「標的」配列と呼ばれ得る。部位特異的様式でDNAを認識し、これに結合することができるポリペプチドドメインは、一般的にドメインが元々単離されたタンパク質以外のポリペプチド中で発現する場合でさえ、正確に折り畳み、独立に機能して部位特異的様式でDNAに結合する。同様に、DNA結合ポリペプチドによる認識および結合のための標的配列は、一般的に大型DNA構造(例えば、染色体)中に存在する場合でさえ、特に標的配列が位置する部位が可溶性細胞タンパク質にアクセス可能であることが知られているもの(例えば、遺伝子)である場合に、このようなポリペプチドによって認識され、結合され得る。
特定の実施形態では、キメラポリペプチドに標的配列との特異的結合を与えるために、標的ヌクレオチド配列を特異的に認識し、これに結合するDNA結合ポリペプチドが、キメラポリペプチドに含まれ得る。例では、これらのポリペプチドは上に記載されているので、このようなキメラポリペプチドは、例えば、限定されないが、ヌクレアーゼ、リコンビナーゼおよび/またはリガーゼポリペプチドを含み得る。DNA結合ポリペプチド、ならびにヌクレアーゼ、リコンビナーゼおよび/またはリガーゼポリペプチドを含むキメラポリペプチドは、他の機能的ポリペプチドモチーフおよび/またはドメイン、例えば、限定されないが、キメラタンパク質中の機能的ポリペプチド間に位置するスペーサー配列;リーダーペプチド;融合タンパク質を小器官(例えば、核)に標的化するペプチド;細胞酵素によって切断されるポリペプチド;ペプチドタグ(例えば、Myc、His等);およびキメラポリペプチドの機能に干渉しない他のアミノ酸配列を含んでもよい。
具体的な実施形態では、キメラポリペプチドが、外因性核酸またはドナーDNAが組み込まれ得る標的化部位特異的二本鎖DNA切断に送達されるよう設計され得るカスタム設計のジンクフィンガーヌクレアーゼ(ZFN)である(参照により本明細書に組み込まれる、共有されている米国特許出願公開第20100257638号明細書参照)。ZFNは、制限エンドヌクレアーゼ(例えば、FokI)からの非特異的切断ドメインと、ジンクフィンガーDNA結合ドメインポリペプチドとを含むキメラポリペプチドである。例えば、Huang et al.(1996) J.Protein Chem.15:481-9;Kim et al.(1997a) Proc.Natl.Acad.Sci.USA 94:3616-20;Kim et al.(1996) Proc.Natl.Acad.Sci.USA 93:1156-60;Kim et al.(1994) Proc Natl.Acad.Sci.USA 91:883-7;Kim et al.(1997b) Proc.Natl.Acad.Sci.USA 94:12875-9;Kim et al.(1997c) Gene 203:43-9;Kim et al.(1998) Biol.Chem.379:489-95;Nahon and Raveh (1998) Nucleic Acids Res.26:1233-9;Smith et al.(1999) Nucleic Acids Res.27:674-81を参照されたい。いくつかの実施形態では、ZFNが非標準ジンクフィンガーDNA結合ドメインを含む(参照により本明細書に組み込まれる、共有されている米国特許出願公開第20080182332号明細書参照)。FokI制限エンドヌクレアーゼは、DNAを切断し、二本鎖切断を導入するために、ヌクレアーゼドメインを介して二量体化しなければならない。結果として、このようなエンドヌクレアーゼからのヌクレアーゼドメインを含むZFNも、標的DNAを切断するためにヌクレアーゼドメインの二量体化を要する。Mani et al.(2005) Biochem.Biophys.Res.Commun.334:1191-7; Smith et al.(2000) Nucleic Acids Res.28:3361-9。ZFNの二量体化は、2つの隣接する正反対に配向したDNA結合部位によって促進され得る。前記。
本発明の実施形態は、少なくとも1つのFAD2座への部位特異的組込みのための外因性核酸、例えば、限定されないが、PTU、ELP、ETIPまたはORF;標的化エンドヌクレアーゼをコードするヌクレオチド配列を含む核酸;および前記のいずれかまたは両方の少なくとも1つを含むベクターからなる群から選択される1つまたはそれ以上の核酸を含んでもよい。したがって、いくつかの実施形態で使用するための特定の核酸は、ポリペプチドをコードするヌクレオチド配列、構造ヌクレオチド配列ならびに/或いはDNA結合ポリペプチド認識および結合部位を含む。
上記のように、例えば、ポリペプチドの発現、突然変異遺伝子の修正または野生型遺伝子の発現増加のために、外因性配列(「ドナー配列」または「ドナー」または「導入遺伝子」とも呼ばれる)が挿入される。ドナー配列が典型的には配置されるゲノム配列と同一ではないことが容易に明らかであるだろう。ドナー配列は、対象となる位置での効率的なHDRを可能にするための相同性の2つの領域が隣接した非相同性配列を含むことができる。さらに、ドナー配列は、細胞クロマチン中の対象となる領域と相同性でない配列を含むベクター分子を含むことができる。ドナー分子は、細胞クロマチンと相同性のいくつかの不連続領域を含むことができる。例えば、対象となる領域中に通常は存在しない配列の標的化挿入のために、前記配列がドナー核酸分子中に存在し、対象となる領域中の配列と相同性の領域が隣接していてもよい。
いくつかの実施形態では、標的化エンドヌクレアーゼをコードするヌクレオチド配列が、標的化エンドヌクレアーゼ中に含まれるポリペプチドをコードする野生のヌクレオチド配列の操作(例えば、ライゲーション)によって操作され得る。例えば、DNA結合ポリペプチドに対応する遺伝子のヌクレオチド配列を同定するために、DNA結合ポリペプチドを含むタンパク質をコードする遺伝子のヌクレオチド配列が調査され得、このヌクレオチド配列がDNA結合ポリペプチドを含む標的化エンドヌクレアーゼをコードするヌクレオチド配列の要素として使用され得る。あるいは、例えば、遺伝暗号の縮重にしたがって、標的化エンドヌクレアーゼをコードするヌクレオチド配列を推定するために、標的化エンドヌクレアーゼのアミノ酸配列が使用され得る。
いくつかの実施形態では、対象となるポリペプチドおよび/または標的化エンドヌクレアーゼをコードする少なくとも1つの外因性ポリヌクレオチド配列を含む少なくとも1つの核酸分子が、発現のために細胞、組織または生物に導入され得る。例えば、少なくとも1つのFAD2座中に含まれるヌクレオチド配列を特異的に認識する標的化エンドヌクレアーゼをコードするポリヌクレオチド配列を含む核酸分子が、標的化エンドヌクレアーゼの発現のために細胞に導入され得、また対象となるポリペプチドをコードするポリヌクレオチド配列が、例えば、発現した標的エンドヌクレアーゼによる座における二本鎖切断の導入後の相同組換えによって少なくとも1つのFAD2座に組み込まれ、対象となるポリペプチドが組み込まれたポリヌクレオチド配列から発現するように、対象となるポリペプチドをコードするポリヌクレオチド配列を含む核酸分子が細胞に導入され得る。
いくつかの実施形態では、少なくとも1つの修飾された(例えば、外因性配列の破壊および/または標的化組込み)FAD2座(例えば、大豆FAD2 2.3座および/またはFAD2 2.6座)を含む植物細胞を含むトランスジェニック植物が提供される。特定の実施形態では、このような植物が、植物組織または植物細胞の形質転換、および全植物の再生によって産生され得る。さらなる実施形態では、このような植物が、部位特異的様式での少なくとも1つのFAD2座における外因性核酸の導入、または修飾FAD2座の生殖質への遺伝子移入を通して得られ得る。このような植物細胞を含む植物材料も提供される。このような植物材料は、植物細胞を含む植物から得られ得る。
ダイズにおいてfad2と連結した(例えば、密に連結した)分子マーカーが提供される。例えば、HO形質(fad2)に関与する配列を含むDNAセグメントが同定される。これらのセグメントは、ゲノム連鎖群中の突然変異対立遺伝子と連結した(例えば、密に連結した)マーカーの周りおよびその間に位置している。したがって、不活性化突然変異を有する突然変異FAD2遺伝子を含む核酸分子も提供される。同定されるセグメント、およびそのマーカーは、一部はダイズゲノムの連鎖群中の位置によって、本主題に含まれる。
シーケンシング反応
ゲノムDNAを大豆組織から単離した。ゲノムDNAを、栽培品種X5およびWestagについては凍結乾燥胚形成浮遊細胞から、また栽培品種Jack、Williams 82およびMaverickについては若葉から単離および精製した。製造業者のプロトコルによって、DNeasy Plant Mini Kit(商標)(Qiagen;Carlsbad、CA)を用いてゲノムDNAを抽出した。
プライマーMA49(配列番号1 caagggttccaaacacaaagcc)およびMA51(配列番号2 catcaatacttgttcctgtacc)、またはMA50(配列番号3 gaagaagcctctctcaagggttc)およびMA51を用いたPCRによって、FAD2 2.3およびFAD2 2.6ゲノムDNA配列を増幅した。1140bpの遺伝子の塩基約40〜1140の断片についてゲノムDNA配列を得た。PCR反応条件は、初期変性について98℃で1分、次いで、35サイクルの98℃で30秒、60℃で15秒、72℃で3分、および72℃で5分の最後の伸長であった。
実施例2:FAD2遺伝子に特異的なジンクフィンガー結合ドメインの設計
ZFN構築物
実施例2に記載されるように、アッセイを用いて同定された、代表的なジンクフィンガーヌクレアーゼのZFN発現構築物を含むプラスミドベクターを、設計し、完成させた。
合成デノボ線状片を高コピープラスミドベクター中に結合することによって、FAD2ドナーベクターを構築した。pDAB115620(図4)およびpDAB115622(図5)を大豆ゲノムのFAD2座へのドナー組込みに使用した。ドナーベクターの両方を、ジンクフィンガーヌクレアーゼ結合ドメインを含むように合成した。pDAB115620(「ドナー1」)は37354:37355ZFN結合ドメイン、37366:37367ZFN結合ドメイン、37370:37371ZFN結合ドメインおよび37374:37375ZFN結合ドメインを含む。pDAB115622(「ドナー2」)は37384:37385ZFN結合ドメイン、37392:37393ZFN結合ドメインおよび37398:37399ZFN結合ドメインを含む。ZFN結合ドメインは対応する発現ジンクフィンガーヌクレアーゼによって認識され、ドナーベクターおよびジンクフィンガーヌクレアーゼベクターを用いた植物細胞の同時形質転換中に切断される。
大豆(例えば、ダイズ栽培品種Maverick)プロトプラスト系形質転換法を開発した。プロトプラストを、葉外植片から産生されたカルスに由来するMaverick懸濁培養液から単離した。下記の技術が本方法を説明する。
大豆細胞懸濁液を、3%(w/v)スクロース、0.5mg/L 2,4−Dおよび7gのbacto agar、pH5.7を含む新鮮なLS培地(Linsmaier and Skoog 1965)への1:5希釈によって7日毎に継代培養した。全ての実験は、下記のプロトコルに基づいて、継代培養7日後に開始して行った。
30mlの継代培養7日後のMaverick懸濁培養液を50ml円錐形遠心管に移し、200gで3分間遠心分離すると、管1本当たり約10mlの沈降細胞体積(settled cell volume)(SCV)が得られた。細胞ペレットを乱すことなく、上清を除去した。20mlの酵溶液(MMG溶液(4mM MES、0.6Mマンニトール、15mM MgCl2、pH6.0)中0.3%ペクトリアーゼ(320952;MP Biomedicals)、3%セルラーゼ(「Onozuka」R10(商標);Yakult Pharmaceuticals、日本))を、懸濁細胞4SCV毎に添加し、管をParafilm(商標)で包んだ。管をプラットホームロッカーに一晩(約16〜18時間)置いた。翌朝、消化細胞のアリコートを顕微鏡的に観察して細胞壁の消化が十分であることを確認した。
細胞/酵素溶液を100μMセルストレーナーを通してゆっくり濾過した。セルストレーナーをW5+培地(1.82mM MES、192mM NaCl、154mM CaCl2、4.7mM KCl、pH6.0)10mlですすいだ。70μMスクリーンを用いて濾過工程を繰り返した。W5+培地10mlを添加することによって、最終体積を40mlにした。管を反転させることによって細胞を混合した。スクロースクッション溶液(500mMスクロース、1mM CaCl2、5mM MES−KOH、pH6.0)8mlを、細胞を含む50ml円錐形遠心管の底部に添加することによって、プロトプラストをスクロースクッション溶液上にゆっくり積層した。管をスインギングバケットローター中350gで15分間遠心分離した。5mlピペットチップを使用してプロトプラストバンド(約7〜8ml)をゆっくり取り出した。次いで、プロトプラストを50ml円錐形管に移し、W5+洗浄液25mlを添加した。管をゆっくり反転させ、200gで10分間遠心分離した。上清を除去し、MMG溶液10mlを添加し、管をゆっくり反転させてプロトプラストを再懸濁した。血球計算器またはフローサイトメーターを用いてプロトプラスト密度を測定した。典型的には、細胞懸濁液4PCVが約200万個のプロトプラストを産生した。
MMGを用いてプロトプラスト濃度を160万個/mlに調整した。300μlのプロトプラストアリコート(約500,000個のプロトプラスト)を2ml滅菌チューブに移した。プロトプラストをチューブに移す間、プロトプラスト懸濁液を規則的に混合した。実験設計にしたがって、プラスミドDNAをプロトプラストアリコートに添加した。プロトプラストのチューブを含むラックを1分間毎に3回ゆっくり反転させてDNAとプロトプラストを混合した。プロトプラストを室温で5分間インキュベートした。300μlのポリエチレングリコール(PEG4000)溶液(40%エチレングリコール(81240−Sigma Aldrich)、0.3Mマンニトール、0.4M CaCl2)をプロトプラストに添加し、チューブのラックを1分間混合し、インキュベーション中に2回穏やかに反転させながら5分間インキュベートした。W5+1mlをチューブにゆっくり添加し、チューブのラックを15〜20回反転させた。次いで、チューブを350gで5分間遠心分離し、ペレットを乱すことなく上清を除去した。WI培地(4mM MES、0.6Mマンニトール、20mM KCl、pH6.0)1mlを各チューブに添加し、ラックを穏やかに反転させてペレットを再懸濁した。ラックをアルミ箔で覆い、横置きして23℃で一晩インキュベートした。
Quanta Flow Cytometer(商標)(Beckman−Coulter Inc.)を用いて、プロトプラストの定量化および形質転換効率を測定した。形質転換約16〜18時間後、各複製物から100μlをサンプリングし、96ウェルプレートに入れ、WI溶液を用いて1:1希釈した。複製物を3回再懸濁し、フローサイトメトリーを用いて100μlを定量化した。サンプルを解析に供する前に、サンプルを200gで5分間遠心分離し、上清を除去し、サンプルを液体窒素で瞬間凍結した。次いで、分子解析のために処理するまで、サンプルを−80℃冷凍庫に入れた。
上記形質転換方法論を用いて、設計ZFNを大豆プロトプラストに形質転換した。FAD2座についての切断効率を、米国仮特許出願第61/736,856号明細書に記載されている座破壊アッセイを介して種々のZFNについて評価した。さらに、イン−アウトPCRアッセイを介してドナー配列のFAD2座へのジンクフィンガーヌクレアーゼ媒介組込みを評価し、得られたPCRアンプリコンをシーケンシングして大豆ゲノムへのドナー組込みを特徴付けた。
座破壊アッセイを用いて標的化実験からのDNAサンプルを解析して、FAD2 ZFN切断部位における修飾を検出またはNHEJによる標的化を評価した。 FAD2標的中の無傷ZFN結合部位を測定するようqPCRアッセイを設計した。ZFN媒介ドナー挿入または切断、引き続いてNHEJ修復が、ZFN結合部位の喪失およびその後の検出可能なqPCRシグナルの減少をもたらす。有意な切断活性を有するZFNは、ドナーのみの処理と比べてシグナルが減少したアンプリコンの産生をもたらした。座破壊アッセイに使用されるプライマーおよびプローブが表4に提供され、FAD2座上のその相対位置が図7に示される。
標的化ドナー挿入を確認するために、全ての処理からのDNAを座特異的イン−アウトPCRアッセイに供した。実験のドナーベクターを、FAD2座への標的化組込みについて試験されている全てのZFNについて結合部位を含むように設計した。ZFNおよびドナーの大豆細胞への同時送達は、標的およびドナーベクターにおけるZFN結合部位の切断、ならびに非相同末端結合機構を介したその後のドナーの切断FAD2座への組込みをもたらす。ZFN切断により産生されたFAD2染色体部位および線状ドナーベクターの末端が、FAD2座への組込み前にプロセシングを受け、不完全な末端結合産物をもたらし得る。標的における標的化組込みの確認を「イン−アウト」PCR戦略に基づいて行った(「Out」プライマーは野生のゲノム座の配列を認識し、「In」プライマーはドナーDNA中の配列に結合する)。挿入ジャンクションの5’末端と3’末端の両方でイン−アウトPCRアッセイを行った。
pDAB1115620およびF2 ZFN2_WT、またはpDAB1115620およびF2 ZFN2_HFを用いて完了したイン−アウトPCR標的化実験の各々からの(予想されるサイズの)アンプリコンの2つをプラスミドにクローニングした。Sangerシーケンシング法を用いて、得られたプラスミドをシーケンシングした。 配列を基準配列(FokI切断から生じると予想される一本鎖4bp末端を複製して末端の全ての可能な組み合わせを表した)にアラインメントした。10個の特有の配列パターンが、得られた23個のクローニング配列から見出された(図8)。全ての配列パターンがZFN結合部位間に位置するFAD2ゲノム基準配列(GAAATTTC)の一部を保持したが、これらの配列パターンはFAD2ゲノム基準配列に対する欠失も有していた。 配列4WT1および4WT4は、GAAATTTC配列の3’末端上のZFN結合部位に広がる欠失を含んでいた。2つの配列、1HF4および6HF4は一塩基挿入を有していた。観察されたDNA配列パターンは、ドナーDNAの大豆FAD2座への標的化が起こったことを証明している。
Claims (11)
- 部位特異的様式で大豆細胞中のFAD2遺伝子中の配列番号14〜配列番号20のいずれか1つで表されるヌクレオチド配列のいずれか内の標的部位をヌクレアーゼにより切断して、それによって前記FAD2遺伝子中に切断部位を生じる工程を含む、大豆細胞のFAD2遺伝子を修飾する方法であって、
少なくとも1つのFAD2遺伝子が前記切断部位に対象となる外因性核酸配列を組み込むことにより改変され、前記FAD2遺伝子が、配列番号4で表されるヌクレオチド配列を含むFAD2 2.3遺伝子またはそのホモログもしくはパラログ、配列番号9で表されるヌクレオチド配列を含むFAD2 2.6遺伝子またはそのホモログもしくはパラログ、またはその両方である、方法。 - 前記FAD2遺伝子が、FAD2 2.3、FAD2 2.6遺伝子、または両方である、請求項1に記載の方法。
- 前記ヌクレアーゼが、DNA結合ドメイン、および切断ドメインまたは切断ハーフドメイン或いは前記ヌクレアーゼをコードするポリヌクレオチドを含み、前記DNA結合ドメインが特異的に前記標的部位に結合し、前記標的部位またはその近くを切断して、それによって前記切断部位を生じる、請求項1または2に記載の方法。
- 前記DNA結合ドメインが、メガヌクレアーゼDNA結合ドメイン、ロイシンジッパーDNA結合ドメイン、転写活性化物質様(TAL)DNA結合ドメイン、RNAガイド化CRISPR−Cas9、リコンビナーゼ、ジンクフィンガータンパク質DNA結合ドメイン、および前記のいずれかのキメラ(chimeric)組み合わせからなる群から選択される、請求項3に記載の方法。
- 前記切断ドメインまたは切断ハーフドメインが、IIS型制限エンドヌクレアーゼからの切断ハーフドメイン、FokIエンドヌクレアーゼからの切断ハーフドメイン、StsIエンドヌクレアーゼからの切断ハーフドメイン、Casタンパク質、およびホーミングエンドヌクレアーゼからなる群から選択される、請求項3または請求項4に記載の方法。
- 前記ヌクレアーゼが、ジンクフィンガーヌクレアーゼ(ZFN)の対を含むジンクフィンガーヌクレアーゼであり、各ジンクフィンガーヌクレアーゼが、F1からF5またはF1からF6に整列された5つまたは6つのジンクフィンガードメインを含むジンクフィンガータンパク質を含み、各ジンクフィンガードメインが、下記表の1つの列(横一列)に整列され、示される認識ヘリックス領域を含む、請求項3から5のいずれかに記載の方法であって、
- 部位特異的様式での前記切断が、配列番号4で表されるヌクレオチド配列を含むFAD2 2.3および配列番号9で表されるヌクレオチド配列を含むFAD2 2.6のいくつかであるが全てではないコピーに特異的である、請求項1から6のいずれかに記載の方法。
- 対象となる前記外因性核酸配列が、標的部位に結合するDNA結合ドメインを含む配列、1つまたはそれ以上の殺虫剤耐性遺伝子、1つまたはそれ以上の除草剤耐性遺伝子、1つまたはそれ以上の窒素利用効率遺伝子、1つまたはそれ以上の水利用効率遺伝子、1つまたはそれ以上の栄養価遺伝子、1つまたはそれ以上のDNA結合遺伝子、1つまたはそれ以上の選択可能なマーカー遺伝子、およびこれらの組み合わせからなる群から選択される、請求項1から7のいずれかに記載の方法。
- FAD2 2.3遺伝子および/またはFAD2 2.6遺伝子の少なくとも1つの1つまたはそれ以上のコピーに組み込まれた前記外因性核酸配列を含むように請求項1から8のいずれかに記載の方法によって修飾された大豆細胞を含む大豆細胞または大豆植物であって、前記FAD2 2.3遺伝子が、配列番号4により表されるヌクレオチド配列またはそのホモログもしくはパラログを含み、前記FAD2 2.6遺伝子が、配列番号9で表されるヌクレオチド配列またはそのホモログもしくはパラログを含む、大豆細胞または大豆植物。
- 前記核酸配列が配列番号14〜配列番号20で表されるヌクレオチド配列からなる群から選択される標的部位またはその近くで組み込まれる、請求項9に記載の細胞または植物。
- 配列番号14〜配列番号20で表されるヌクレオチド配列からなる群から選択される核酸標的部位またはその近くを切断する、FAD2遺伝子を修飾するために使用するための部位特異的ジンクフィンガーヌクレアーゼであって、
前記FAD2遺伝子が、配列番号4で表されるヌクレオチド配列を含むFAD2 2.3遺伝子、配列番号9で表されるヌクレオチド配列を含むFAD2 2.6遺伝子、またはその両方であり、前記ジンクフィンガーヌクレアーゼが、ジンクフィンガーヌクレアーゼの対を含み、各ジンクフィンガーヌクレアーゼがF1からF5またはF1からF6に整列される5または6個のジンクフィンガードメインを含み、各ジンクフィンガードメインが認識ヘリックス領域を含み、前記ジンクフィンガータンパク質が、以下の表の一つの列(横一列)に整列され、示される前記認識ヘリックス領域を含み、
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US61/697,886 | 2012-09-07 | ||
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