JP2018530348A - 植物プロトプラストからゲノム編集植物体を高効率で製造する方法 - Google Patents
植物プロトプラストからゲノム編集植物体を高効率で製造する方法 Download PDFInfo
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Abstract
Description
前記方法において、(i)ステップは、分離された植物プロトプラストにCasタンパク質およびガイドRNAを導入して前記植物プロトプラストのゲノムを編集するステップとして、標的遺伝子をコードするDNAに特異的なガイドRNAとCasタンパク質を植物プロトプラストに導入して、外来DNAのゲノムへの挿入を最小化し、且つ植物プロトプラストを短時間内に形質転換させることができる。
レタスプロトプラストを分離するために、レタスのチョンチマ(Cheongchima)種子を70%のエタノール、0.4%のヒポクロリット(hypochlorite)溶液で15分間滅菌した後、蒸留水で3回洗浄し、2%のスクロースを含有する1/2X MS固形培地で培養した。培養は、成長室で16時間明(150μmol/m2s)、8時間暗条件で25℃で行った。培養7日目のレタス子葉を、酵素溶液(1.0%のセルラーゼR10、0.5%のマセロザイム(macerozyme)R10、0.45Mのマンニトール、20mMのMES[pH5.7]、CPW溶液)とともに暗い状態で25℃で12時間、40rpmで撹拌しながらインキュベーションして分解した後、同量のW5溶液で希釈させた。前記混合物を濾過させ、丸底チューブで100gで5分間遠心分離してプロトプラストを回収した。再懸濁されたプロトプラストをCPW 21S溶液(21%[w/v]スクロース含有CPW溶液、pH5.8)に浮遊させて精製し、80gで7分間遠心分離した。精製されたプロトプラストをW5溶液で洗浄した後、70gで5分間遠心分離してペレットを製作した。最後に、プロトプラストをW5溶液に再懸濁し、ヘマサイトメータ(hemacytometer)を用いて顕微鏡で数を数えた。
形質転換の前に、Cas9タンパク質を含む保存緩衝液(20mMのHEPES,pH7.5,150mMのKCl,1mMのDTT,および10%のグリセロール)をsgRNAと混合し、常温で10分間インキュベーションした。
レタスプロトプラストの再生のために、RNPで形質転換させたプロトプラストを、375mg/LのCaCl2・2H2O、18.35mg/LのNaFe‐EDTA、270mg/Lのコハク酸ナトリウム(sodium succinate)、103g/Lのスクロース、0.2mg/Lの2,4‐D、0.3mg/Lの6‐BAP(benzylaminopurine)、および0.1g/LのMESを含む1/2X B5培養培地に再懸濁させた。次に、プロトプラストを、1/2X B5培地と2.4%のアガロースとの1:1溶液とともに混合し、2.5x105プロトプラスト/mlの密度で培養した。アガロースにより囲まれたプロトプラストを6‐ウェルプレートに移し、2mlの1/2X B5培養培地で25℃の暗条件で培養した。7日後、前記培地を新しい培地に入れ替え、25℃の明条件(16時間明[30μmol/m−2s−1]および8時間暗条件)で培養した。培養3週後、ミクロカリを数mmの直径まで育て、30g/Lのスクロース、0.6%のプラントアガー、0.1mg/Lのα‐NAA(naphthalaneacetic acid)、0.5mg/LのBAPを含有するMS再生培地に移して培養した。再生培地で約4週経過した後、多数のレタスの芽が誘導されたことが観察された。
RGEN‐RNPを適用したプロトプラストまたは再生されたカルスのゲノムDNAで標的位置(on‐target site)を増幅させた。インデックスとシーケンシングアダプタを追加的なPCRにより付加した。Illumina Miseq(v2、300‐cycle)を用いてハイスループットシーケンシング(high‐throughput sequencing)を行った。用いられたプライマーは下記表1に示した。
DNeasy Plant Mini Kit(Qiagen)を用いて、プロトプラストまたはカルスからゲノムDNAを分離した。標的DNA領域を増幅させてT7E1分析を行った。具体的に、PCR産物を95℃に変性させ、サーマルサイクラー(thermal cycler)を用いて常温まで温度をゆっくりと低めた。アニーリングされたPCR産物を37℃で20分間T7エンドヌクレアーゼI(ToolGen,Inc.)とインキュベーションし、アガロースゲル電気泳動により分析した。
1X NEB緩衝液3で、PCR産物(300‐400ng)、Cas9タンパク質(1μg)、およびsgRNA(750ng)を反応体積10μlで、37℃で60分間インキュベーションした。次に、前記反応混合物にRNase A(4μg)を添加し、37℃で30分間インキュベーションしてsgRNAを除去した。次に、6X停止溶液(30%のグリセロール、1.2%のSDS、および250mMのEDTA)を添加して前記反応を停止させた。2.5%のアガロースゲルを用いてDNA産物を電気泳動した。
本発明者らは、レタスにおいてBR(brassinosteroid)信号伝逹経路で負の制御因子をコードするBIN2(brassinosteroid intensive 2)遺伝子を欠損させるためのRGEN(RNA‐guided engineered nuclease)標的位置を設計した(図1のa、配列番号11)。次に、PEG(polyethylene glycol)の存在下でレタスから分離したプロトプラストにRGEN RNP(ribonucleoprotein)を形質転換させ、T7E1(T7 endonuclease 1)分析および標的化ディープシーケンシング(targeted deep sequencing)により、RGENによって引き起こされた遺伝子変異を測定した。その結果、インデル(insertion and deletion、indel)は、予想された位置、すなわち、NGG PAM(protospacer‐adjacent motif)の上流の3nt(nucleotide)位置で、T7E1分析で8.3%〜11%、NGS分析で3.2%〜5.7%の頻度で現われた(図1のbおよびc)。
実験的誤差を排除し、且つ実験例1の現象を確認するために、本発明者らは、キャベツ(Brassica oleracea)で追加的に独立した実験を行った。実験例1で標的として用いられたBIN2遺伝子の欠損が、プロトプラストの再生過程で成長および生存性に影響を与えた可能性を排除できないため、本発明者らは、プロトプラストまたはカルスの成長および生存性とは無関係な遺伝子を選別しようとした。そこで、グルコシノレート(glucosinolate)経路で側鎖変異に影響を与えるタンパク質をコードするキャベツ(B.oleraceae)のGSL―ALK(Glucosinolate‐oxoglutarate‐dependent dioxygenase homolog)遺伝子を標的とし、それを欠損させるために7個のsgRNA配列を設計した(表2)。
Claims (22)
- (i)分離された植物プロトプラストにCasタンパク質およびガイドRNAを導入して前記植物プロトプラストのゲノムを編集するステップと、
(ii)前記植物プロトプラストを再生させてゲノム編集植物体を製造するステップと、
を含む、植物プロトプラストから製造されるゲノム編集植物体の製造効率を増加させる方法。 - 前記ガイドRNAは、標的遺伝子をコードするDNAに特異的なものであることを特徴とする請求項1に記載の方法。
- 前記標的遺伝子は、BIN2(brassinosteroid intensive 2)遺伝子またはGSL‐ALK(Glucosinolate‐oxoglutarate‐dependent dioxygenase homolog)遺伝子であることを特徴とする請求項2に記載の方法。
- 前記ゲノム編集は、ノックアウト(knock−out)またはノックイン(knock−in)により行われることを特徴とする請求項1に記載の方法。
- 前記ガイドRNAは、crRNAおよびtracrRNAを含むデュアルRNA(dual RNA)または単一鎖ガイドRNA(sgRNA)の形態であることを特徴とする請求項1に記載の方法。
- 前記単一鎖ガイドRNAは、crRNAおよびtracrRNAの一部分を含むことを特徴とする請求項5に記載の方法。
- 前記ガイドRNAは、裸のRNA(naked RNA)の形態であることを特徴とする請求項1に記載の方法。
- 前記Casタンパク質は、Cas9タンパク質またはこれの変異体であることを特徴とする請求項1に記載の方法。
- 前記Casタンパク質は、NGGトリヌクレオチドを認識することを特徴とする請求項1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)に連結されていることを特徴とする請求項1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で置き換えられたCas9タンパク質の突然変異形態であることを特徴とする請求項8に記載の方法。
- 前記アミノ酸は、アラニン(alanine)であることを特徴とする請求項11に記載の方法。
- 前記Casタンパク質は、ストレプトコッカス(Streptococcus)属由来のものであることを特徴とする請求項1に記載の方法。
- 前記ストレプトコッカス(Streptococcus)属は、ストレプトコッカス・ピオゲネス(Streptococcus pyogenes)であることを特徴とする請求項13に記載の方法。
- 前記植物プロトプラストは、レタス(Lactuca sativa)またはキャベツ(Brassica oleracea)由来のものであることを特徴とする請求項1に記載の方法。
- 前記導入は、同時形質移入(co‐transfection)または段階的形質移入(serial‐transfection)の形態で行われることを特徴とする請求項1に記載の方法。
- 前記段階的形質移入は、Casタンパク質またはCasタンパク質をコードする核酸を形質移入した後に、裸のガイドRNA(naked guide RNA)を形質移入することで行われることを特徴とする請求項16に記載の方法。
- 前記導入は、マイクロインジェクション法、電気穿孔法、DEAE−デキストラン処理法、リポフェクション、ナノ粒子−媒介形質移入、タンパク質伝達ドメイン−媒介形質導入およびPEG−媒介形質移入からなる群から選択される方法によって行われることを特徴とする請求項1に記載の方法。
- 前記再生させるステップは、ゲノム編集された植物プロトプラストを培養してカルス(callus)を形成するステップと、前記カルスを追加的に培養することで、再生された植物体を製造するステップと、を含むことを特徴とする請求項1に記載の方法。
- 請求項1〜19のいずれかに記載の方法によって製造されたゲノム編集植物プロトプラストから再生された植物。
- 標的遺伝子をコードするDNAに特異的なガイドRNAおよびCasタンパク質を含む、植物プロトプラストから製造されるゲノム編集植物体の製造効率を増加させるための組成物。
- 前記標的遺伝子は、BIN2(brassinosteroid intensive 2)遺伝子またはGSL‐ALK(Glucosinolate‐oxoglutarate‐dependent dioxygenase homolog)遺伝子であることを特徴とする請求項21に記載の組成物。
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