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METHOD FOR PRODUCING FUSION PROTEIN, NUCLEIC ACID, CELL, AND ANIMAL

Foreign code F200010018
File No. (S2018-0543-N0)
Posted date Jan 30, 2020
Country WIPO
International application number 2019JP020244
International publication number WO 2019225638
Date of international filing May 22, 2019
Date of international publication Nov 28, 2019
Priority data
  • P2018-101060 (May 25, 2018) JP
Title METHOD FOR PRODUCING FUSION PROTEIN, NUCLEIC ACID, CELL, AND ANIMAL
Abstract A fusion protein including a Cas9 protein and a modified peptide that modifies the Cas9 protein, wherein the modified peptide includes: a peptide composed of the amino acid sequence described in SEQ ID NO: 29; or a peptide composed of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence described in SEQ ID NO: 29, the peptide having activity to localize the Cas9 protein in the nucleus by forming a fusion protein with the Cas9 protein.
Outline of related art and contending technology BACKGROUND ART
And disable a particular gene knockout (hereinafter, referred to as a 'KO'.) A mouse or a particular gene locus of an exogenous gene knock and (hereinafter, referred to as' KI '.) The mouse, the function of the gene in vivo can be evaluated directly, many medical, life science research have been used.
Conventional, a mouse or a KO mice KI, embryonic stem (Embryonic Stem, hereinafter, referred to as' ES '.) Using the cells have been produced by a gene targeting. However, its manufacturing is the cost of a large amount of time and year was a problem. To solve this problem is, genome editing technique.
In the genome editing, only knows the particular DNA sequence, an artificial restriction enzyme cutting site of the cell can be introduced, the target gene can be KO. In addition, introduction of foreign genes in the vicinity of the cleavage site sandwiched between the donor DNA sequence, an artificial restriction enzymes can be introduced into the cell at the same time, the target loci may be foreign genes in the KI.
As artificial restriction enzymes can be used for genome editing, at the present time is the most potent Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated Protein 9 (hereinafter 'CRISPR-Cas9' may be referred to.) System.
Among CRISPR-Cas9, Streptococcus pyogenes (Streptococcus pyogenes, hereinafter, referred to as' Sp '.) Derived from CRISPR-Cas9(hereinafter, referred to as' SpCas9 '.) Is a simple and efficient to have the ability to cut DNA for, many studies have been used.
Sp-CRISPR-Cas9 system, mainly, has a function to recognize the single guide RNA target sequence (hereinafter, referred to as' sgRNA '.) And, the target sequence has a main function in a complex protein RNA - Cas9 protein. And a mouse simultaneously fertilized eggs Cas9 sgRNA of these proteins by introducing, KO mice can be obtained.
In addition, using one of 2 different sgRNA, upstream and downstream of the target gene region by cutting at the same time, the area can be excised from the chromosome can be (KO-cut). In this step, non-homologous end joining error-prone (non-homologous end-joining, hereinafter, referred to as' NHEJ '.) Are used. By this method, it is difficult in a conventional gene targeting method, several hundred million base pairs becomes possible to cut.
In addition, protein and DNA sgRNA Cas9 fertilized eggs at the same time by introducing a donor, the donor site template DNA homologous recombination repair (Homology-directed Repair, hereinafter referred to as' HDR '.) Are repaired by, KI mouse can be obtained.
From the prior studies, HDR activity is not seen in stage G1, an abrupt increase of the S phase, G2/M phase has been reduced by. In addition, the activity NHEJ, the entire cell cycle are clearly observed. Therefore, the presence of the protein in the cell cycle specific Cas9 control, by insertion or deletion mutation NHEJ original purpose is to reduce the amount of introduced (Indel) has been reviewed.
For example, human G1 protein can be degraded during Geminin has been known. In addition, human S/G2 protein is being degraded during Cdt1 has been known. Then, the non-patent document 1, is connected to a portion of the protein Geminin Cas9 human fusion protein (hereinafter, 'SpCas9-hGem' may be referred to.), And, connected to a portion of the protein Cdt1 Cas9 human fusion protein (hereinafter, 'SpCas9-hCdt1' may be referred to.) Was produced and described.
In addition to the non-patent document 1, and G1 is degraded during SpCas9-hGem, SpCas9-hCdt1 and S/G2 is decomposed into phase and according to a (non-patent document 1, such as Fig.1C).
Non-Patent Document 1 also includes, at a locus DNMT3B, 17.1% and SpCas 9 is normal by the KI efficiency, the efficiency by 16.5% and the KI SpCas9-hGem, KI efficiency was by SpCas9-hCdt1 9.9% have been described (non-patent document 1, such as Fig.2C).
Scope of claims (In Japanese)[請求項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。

[請求項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、請求項1に記載の融合タンパク質。

[請求項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G 1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。

[請求項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、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5]
 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6]
 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。

[請求項7]
 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8]
 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。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • UNIVERSITY OF TSUKUBA
  • Inventor
  • MIZUNO Seiya
  • SUGIYAMA Fumihiro
  • TAKAHASHI Satoru
  • MIZUNO Saori
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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