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

外国特許コード F200010018
整理番号 (S2018-0543-N0)
掲載日 2020年1月30日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2019JP020244
国際公開番号 WO 2019225638
国際出願日 令和元年5月22日(2019.5.22)
国際公開日 令和元年11月28日(2019.11.28)
優先権データ
  • 特願2018-101060 (2018.5.25) JP
発明の名称 (英語) METHOD FOR PRODUCING FUSION PROTEIN, NUCLEIC ACID, CELL, AND ANIMAL NEW
発明の概要(英語) 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.
従来技術、競合技術の概要(英語) 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).
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • UNIVERSITY OF TSUKUBA
  • 発明者(英語)
  • MIZUNO Seiya
  • SUGIYAMA Fumihiro
  • TAKAHASHI Satoru
  • MIZUNO Saori
国際特許分類(IPC)
指定国 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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