JPWO2019225638A1 - 融合タンパク質、核酸、細胞及び動物の製造方法 - Google Patents
融合タンパク質、核酸、細胞及び動物の製造方法 Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Abstract
Description
[1]Cas9タンパク質と、前記Cas9タンパク質を修飾する修飾ペプチドとを含む融合タンパク質であって、前記修飾ペプチドは、配列番号29に記載のアミノ酸配列からなるペプチド、又は配列番号29に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ前記Cas9タンパク質との融合タンパク質を形成することにより前記Cas9タンパク質を核に局在させる活性を有するペプチド、を含む、融合タンパク質。
[2]前記修飾ペプチドは、配列番号1に記載のアミノ酸配列からなるペプチド、又は配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ前記Cas9タンパク質との融合タンパク質を形成することにより前記Cas9タンパク質を核に局在させる活性を有するペプチド、を含む、[1]に記載の融合タンパク質。
[3]前記修飾ペプチドが、配列番号2に記載のアミノ酸配列からなるペプチド、又は配列番号2に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ前記Cas9タンパク質との融合タンパク質を形成することにより前記Cas9タンパク質をG1期に分解させる活性を有するペプチド、を更に含む、[1]又は[2]に記載の融合タンパク質。
[4]前記修飾ペプチドが、前記Cas9タンパク質のN末端、C末端又はN末端若しくはC末端以外の部位に配置されている、[1]〜[3]のいずれかに記載の融合タンパク質。
[5][1]〜[4]のいずれかに記載の融合タンパク質をコードする核酸。
[6][1]〜[4]のいずれかに記載の融合タンパク質を細胞中のゲノムDNAに接触させることを含む、ゲノムDNAが編集された細胞の製造方法。
[7]前記細胞が受精卵である、[6]に記載の製造方法。
[8][1]〜[4]のいずれかに記載の融合タンパク質を受精卵中のゲノムDNAに接触させ、ゲノムDNAが編集された受精卵を得ることと、前記ゲノムDNAが編集された受精卵を個体に成長させ、ゲノムDNAが編集された動物を得ることと、を含む、ゲノムDNAが編集された動物の製造方法。
1実施形態において、本発明は、Cas9タンパク質と、前記Cas9タンパク質を修飾する修飾ペプチドとを含む融合タンパク質であって、前記修飾ペプチドは、配列番号1に記載のアミノ酸配列からなるペプチド、又は配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ前記Cas9タンパク質との融合タンパク質を形成することにより前記Cas9タンパク質を核に局在させる活性を有するペプチド、を含む、融合タンパク質を提供する。
1実施形態において、本発明は、上述した融合タンパク質をコードする核酸を提供する。本実施形態の核酸を細胞内に導入することにより、上述した融合タンパク質を発現させることができる。また、本実施形態の核酸を試験管内転写反応系で転写することにより、上述した融合タンパク質をコードするmRNAを得ることができる。
1実施形態において、本発明は、上述した融合タンパク質を細胞中のゲノムDNAに接触させることを含む、ゲノムDNAが編集された細胞の製造方法を提供する。
1実施形態において、本発明は、上述した融合タンパク質を受精卵中のゲノムDNAに接触させ、ゲノムDNAが編集された受精卵を得ることと、前記ゲノムDNAが編集された受精卵を個体に成長させ、ゲノムDNAが編集された動物を得ることと、を含む、ゲノムDNAが編集された動物の製造方法を提供する。
(マウス)
C57BL/Jマウスは日本チャールスリバー社より購入した。マウス作製に使用した偽妊娠マウスとしてはICRマウス(日本チャールスリバー社)を用いた。マウスは、室温23.5℃±2.5℃、湿度52.5°±12.5%、明期14時間:暗期10時間の明暗周期の環境下で飼育した。
12〜20週齢の雌のC57BL/6Jマウスに5unitの妊馬血清性性腺刺激ホルモンを皮下投与し、その48時間後に5unitのヒト絨毛性ゴナドトロピンを腹腔内投与し、雄のオスC57BL/6Jマウスと交配させた。膣栓の存在により交配を確認し、卵管より受精卵(前核期)を採取した。なお、凍結受精卵や体外受精卵は使用しなかった。pX330ベクター、pX330−mGベクター、pX330−mCベクターは滅菌蒸留水で5ng/μLに希釈し、ポアサイズ0.22μmのPVDF膜で濾過して使用した。各種ドナーベクターは滅菌蒸留水で10ng/μLに希釈し、ポアサイズ0.22μmのPVDF膜で濾過して使用した。各ベクターは前核期胚の雄性前核に顕微注入(マイクロインジェクション)した。顕微注入の15分から2時間後に生存していた受精卵を偽妊娠マウスの卵管に移植し、個体へと発生させた。
0.5mm以下のマウスの尾の先端からゲノム抽出機器のPI−200を用いてゲノムDNAを抽出・精製し、遺伝型の解析に用いた。KIアレル及びfloxアレルの確認は、PrimeSTAR GXL DNA Polymerase(TAKARA社)を用いたPCRにより行なった。KOアレルの確認は、AmpliTaq Gold 360 Master Mix(サーモフィッシャーサイエンティフィック社)を用いたPCRにより行なった。
(Cas9融合タンパク質発現ベクターの作製1)
pX330(Plasmid #42230、Addgene社)は、sgRNAとCas9の双方を発現させることができるベクターである。図1(c)の上段にpX330ベクターの構造を示す。pX330ベクターでは、U6プロモーターによりsgRNAが発現する。また、CBhプロモーターによりSpCas9タンパク質が発現する。SpCas9タンパク質のN末端及びC末端にはSV40ウイルス由来の核移行シグナル(NLS)が連結されている。また、N末端側のNLSの更にN末端側に3×FLAGタグペプチドが連結されている。
(Cas9、Cas9−mG、Cas9−mCの細胞周期依存性と細胞内局在の検討)
マウス受精卵を用いて、実験例1で作製した発現ベクターの導入により発現するCas9−mGタンパク質及びCas9−mCタンパク質の細胞周期依存性と細胞内局在を検討した。
(Cas9−mCを用いた大規模ゲノム欠損マウスの作製1)
Cas9−mCにより、大規模なゲノム領域の切除効率が上昇するか否かについて検討した。
(Cas9−mCを用いた大規模ゲノム欠損マウスの作製2)
Cas9−mCにより、大規模なゲノム領域の切除効率が上昇するか否かについて検討した。
(Cas9−mCを用いたノックインマウスの作製1)
受精卵のゲノム編集では、高いGC含量を有する塩基配列からなる遺伝子断片をノックインしたマウスの作出や、同時的に2箇所にLoxP配列を導入しなくてはいけないfloxマウスの作出は難しいことが知られている。これらの高難易度のノックインマウス又はfloxマウス作製にCas9−mCが有用かどうかを検討した。
(Cas9−mCを用いたノックインマウスの作製2)
続いて、Prdm14遺伝子の第6エクソンの上流及び下流にLoxP配列を導入し、Prdm14 floxマウスを作製した。
(Cas9、Cas9−mG、Cas9−mCの細胞内局在の検討)
ヒト胎児腎由来の細胞であるHEK293T細胞を用いて、実験例1で作製した発現ベクターの導入により発現するCas9−mGタンパク質及びCas9−mCタンパク質の細胞内局在を検討した。
(Cas9融合タンパク質発現ベクターの作製2)
実験例1で作製したpX330−mCベクターから発現されるSpCas9タンパク質は、SpCas9タンパク質のN末端及びC末端にSV40ウイルス由来の核移行シグナル(NLS)を有していた。図8(a)に、pX330−mCベクターの構造を示す。
(Cas9融合タンパク質の細胞内局在の検討1)
ヒト胎児腎由来の細胞であるHEK293T細胞を用いて、実験例8で作製した発現ベクターの導入により発現する各融合タンパク質の細胞内局在を検討した。
(Cas9融合タンパク質の細胞内局在の検討2)
実験例9で各融合タンパク質を発現させたHEK293T細胞から、市販のキット(コード番号「295−73901」、和光純薬工業)を用いて可溶性核タンパク質画分及び細胞質タンパク質画分をそれぞれ抽出し、ウエスタンブロッティング法により各Cas9融合タンパク質を検出した。
(Cas9融合タンパク質の分解性の検討)
ヒト胎児腎由来の細胞であるHEK293T細胞を用いて、実験例8で作製したCas9−pFmC融合タンパク質及びCas9−pNmC融合タンパク質の分解性を検討した。
Claims (8)
- Cas9タンパク質と、前記Cas9タンパク質を修飾する修飾ペプチドとを含む融合タンパク質であって、
前記修飾ペプチドは、配列番号29に記載のアミノ酸配列からなるペプチド、又は配列番号29に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ前記Cas9タンパク質との融合タンパク質を形成することにより前記Cas9タンパク質を核に局在させる活性を有するペプチド、を含む、
融合タンパク質。 - 前記修飾ペプチドは、配列番号1に記載のアミノ酸配列からなるペプチド、又は配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ前記Cas9タンパク質との融合タンパク質を形成することにより前記Cas9タンパク質を核に局在させる活性を有するペプチド、を含む、請求項1に記載の融合タンパク質。
- 前記修飾ペプチドが、配列番号2に記載のアミノ酸配列からなるペプチド、又は配列番号2に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなり、かつ前記Cas9タンパク質との融合タンパク質を形成することにより前記Cas9タンパク質をG1期に分解させる活性を有するペプチド、を更に含む、請求項1又は2に記載の融合タンパク質。
- 前記修飾ペプチドが、前記Cas9タンパク質のN末端、C末端又はN末端若しくはC末端以外の部位に配置されている、請求項1〜3のいずれか一項に記載の融合タンパク質。
- 請求項1〜4のいずれか一項に記載の融合タンパク質をコードする核酸。
- 請求項1〜4のいずれか一項に記載の融合タンパク質を細胞中のゲノムDNAに接触させることを含む、ゲノムDNAが編集された細胞の製造方法。
- 前記細胞が受精卵である、請求項6に記載の製造方法。
- 請求項1〜4のいずれか一項に記載の融合タンパク質を受精卵中のゲノムDNAに接触させ、ゲノムDNAが編集された受精卵を得ることと、
前記ゲノムDNAが編集された受精卵を個体に成長させ、ゲノムDNAが編集された動物を得ることと、
を含む、ゲノムDNAが編集された動物の製造方法。
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