JP2018530350A - プロトプラストからの全植物体の製造方法 - Google Patents
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Abstract
【選択図】図4a
Description
[発明を実施するための形態]
核位置信号が接続されてCas9タンパク質はToolGen、Inc.(South Korea)から購入した。Phusionポリメラーゼ(表1−4)を使用したオリゴ−延長(oligo−extension)を介してガイドRNA転写の鋳型を製造した。T7 RNAポリメラーゼ(New England Biolabs)を使用したラン−オフ(run−off)反応でガイドRNAsを試験管内で転写した。反応混合物は、1X DNase I反応バッファーに含まれているDNase I(New England Biolabs)で処理した。転写されたsgRNAsの品質を評価するためにSYBR gold染色(Invitrogen)された8%変性尿素−ポリアクリルアミドゲル(urea−polyacryl amide gel)でsgRNAsを確認した。転写されたsgRNAsをMGTM PCR Product Purification SV(Macrogen)で精製し、分光光度計でsgRNAsの量を測定した。
Claims (32)
- 植物プロトプラスト(protoplast)の一つ以上の内在的遺伝子をノックアウトまたはノックインする段階を含む、植物プロトプラストから植物体を製造する方法。
- 前記植物の内在的遺伝子は、前記ノックアウトまたはノックインによりストレス抵抗性を増加させる遺伝子である、請求項1に記載の方法。
- 前記植物の内在的遺伝子は、植物のブラシノステロイド(brassinosteroid)シグナル伝達に関与する遺伝子である、請求項1に記載の方法。
- (i)前記ノックアウトの段階において、内在的遺伝子はBIN2遺伝子、BKI1遺伝子、及びこれらのホモログ遺伝子からなる群から選択される1つ以上であり、
(ii)前記ノックインの段階において、内在的遺伝子はBRI1遺伝子、BSU遺伝子、BZR1遺伝子、DWF4遺伝子、CYP85A1、およびこれらのホモログ遺伝子からなる群から選択される1つ以上である、請求項1に記載の方法。 - 前記遺伝子のノックアウトは、BIN2遺伝子、BKI1遺伝子、及びこれらのホモログ遺伝子からなる群から選択される1つ以上の遺伝子のアレルの中いずれか一つまたは二つをノックアウトすることである、請求項1に記載の方法。
- 前記遺伝子のノックアウトは、遺伝子ノックアウトにより実行され、前記遺伝子のノックインは、遺伝子ノックインにより実行される、請求項1に記載の方法。
- 前記遺伝子のノックアウトは、BIN2遺伝子、BKI1遺伝子、及びこれらのホモログ遺伝子からなる群から選択される1つ以上の遺伝子に特異的な人工ヌクレアーゼ(engineered nuclease)を利用して実行される、請求項1に記載の方法。
- 前記人工ヌクレアーゼは、ジンクフィンガーヌクレアーゼ(ZFN)、転写アクチベーター様エフェクターヌクレアーゼ(TALEN)、およびRNAガイド人工ヌクレアーゼ(RGEN)からなる群から選択される、請求項7に記載の方法。
- 前記RGENは、BIN2遺伝子、BKI1遺伝子、及びこれらのホモログ遺伝子からなる群から選択される1つ以上の遺伝子の特定配列に特異的に結合するガイドRNA、または前記ガイドRNAをコードするDNA、並びにCasタンパク質をコードする核酸またはCasタンパク質を含む、請求項8に記載の方法。
- 前記遺伝子のノックアウトは、BIN2遺伝子、BKI1遺伝子、及びこれらの同族体ホモログ遺伝子からなる群から選択される1つ以上の遺伝子の特定配列に特異的に結合するガイドRNAまたは前記ガイドRNAをコードするDNA;並びにCasタンパク質をコードする核酸またはCasタンパク質を、植物プロトプラストに導入することで実行される、請求項1に記載の方法。
- 前記ガイドRNAはcrRNAとtracrRNAを含む二重RNA(dual RNA)または単一鎖ガイドRNA(sgRNA)の形態である、請求項10に記載の方法。
- 前記単一鎖ガイドRNAはcrRNA及びtracrRNAの一部を含む、請求項11に記載の方法。
- 前記単一鎖ガイドRNAは、単離されたRNAの形態である、請求項10に記載の方法。
- 前記ガイドRNAをコードするDNAはベクターにコードされており、前記ベクターは、ウイルスベクター、プラスミドベクター、またはアグロバクテリウムベクターである、請求項10に記載の方法。
- 前記Casタンパク質はCas9タンパク質またはその変異体である、請求項10に記載の方法。
- 前記Casタンパク質は、NGGトリヌクレオチド(trinucleotide)を認識する、請求項10に記載の方法。
- 前記Casタンパク質は、タンパク質伝達ドメイン(protein transduction domain)に接続されている、請求項10に記載の方法。
- 前記Cas9タンパク質の変異体は、触媒アスパラギン酸(aspartate)残基が他のアミノ酸に置換されたCas9の変異体である、請求項15に記載の方法。
- 前記アミノ酸は、アラニン(alanine)である、請求項18に記載の方法。
- 前記Casタンパク質をコードする核酸またはCasタンパク質は、ストレプトコッカス属(genus Streptococcus)由来のものである、請求項10に記載の方法。
- 前記ストレプトコッカス属はストレプトコッカスピヨジェンス(Streptococcus pyogenes)である、請求項20に記載の方法。
- 前記植物プロトプラストはレタス(Lactuca sativa)由来のものである、請求項1に記載の方法。
- 前記導入はCasタンパク質をコードする核酸またはCasタンパク質、及び前記ガイドRNAまたはガイドRNAをコードするDNAを、植物プロトプラストに同時形質導入する(co−transfecting)または連続形質導入(serial−transfecting)するものである、請求項10に記載の方法。
- 前記連続形質導入は、Casタンパク質またはCasタンパク質をコードする核酸を初めに形質導入した後、ネイキッドガイドRNA(naked guide RNA)を二番目に形質導入することで実行される、請求項23に記載の方法。
- 前記導入は、微細注入法、電気穿孔法、DEAE−デキストラン処理、リポフェクション、ナノパーティクル−媒介性形質導入、タンパク質形質導入ドメイン媒介性形質導入、およびPEG−媒介形質導入からなる群から選択される方法で実行される、請求項10に記載の方法。
- 遺伝子がノックアウトされた植物プロトプラストを再生させる段階をさらに含む、請求項1に記載の方法。
- 前記再生させる段階は、BIN2遺伝子、BKI1遺伝子、及びこれらのホモログ遺伝子からなる群から選択される1つ以上の遺伝子がノックアウトされた植物プロトプラストを、アガロースを含む培地中で培養してカルス(callus)を形成させる段階、及び前記カルスを再生培地で培養する段階を含む、請求項26に記載の方法。
- 請求項1〜27のいずれか一項に記載の方法によって調製されたゲノム編集された植物プロトプラストから再生された植物。
- BIN2(brassinosteroid Insensitive 2)遺伝子、BKI1遺伝子またはこれらのホモログをコードするDNAに特異的なガイドRNA、または前記ガイドRNAをコードするDNA;並びにCasタンパク質をコードする核酸またはCasタンパク質を含む、植物細胞においてBIN2遺伝子をコードするDNAを切断するための組成物。
- 植物細胞で標的変異(targeted mutagenesis)を誘導する、請求項29に記載の組成物。
- BIN2(brassinosteroid Insensitive 2)遺伝子、BKI1遺伝子またはこれらのホモログをコードするDNAに特異的なガイドRNAまたは前記ガイドRNAをコードするDNA;並びにCasタンパク質をコードする核酸またはCasタンパク質を含む、植物プロトプラストから植物体を製造するための組成物。
- 請求項29〜30のいずれか一項に記載の組成物を含む、植物プロトプラストから植物体を製造するためのキット。
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PCT/KR2016/011217 WO2017061806A1 (en) | 2015-10-06 | 2016-10-06 | Method for producing whole plants from protoplasts |
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EP4178344A1 (en) | 2020-07-13 | 2023-05-17 | The Regents of the University of California | Plant metabolite-mediated induction of biofilm formation in soil bacteria to increase biological nitrogen fixation and plant nitrogen assimilation |
KR102304761B1 (ko) * | 2020-07-15 | 2021-09-28 | 한국생명공학연구원 | StSR4 유전자 교정에 의해 병 저항성이 증가된 감자 식물체의 제조방법 및 상기 방법에 의해 제조된 병 저항성이 증가된 유전체 교정 감자 식물체 |
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KR102280955B1 (ko) * | 2021-06-07 | 2021-07-23 | 전남대학교산학협력단 | 토마토 열매의 아스코르브산 함량을 조절하는 토마토 유래 apx4 유전자 및 이의 용도 |
CN112941077B (zh) * | 2021-02-23 | 2021-09-07 | 东北林业大学 | 靶向敲除GLK基因的CRISPR/Cas9***及其应用 |
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