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METHOD FOR PRODUCING GENE KNOCK-IN CELLS

外国特許コード F180009356
整理番号 (S2016-0981-N0)
掲載日 2018年4月19日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2017JP027838
国際公開番号 WO 2018030208
国際出願日 平成29年8月1日(2017.8.1)
国際公開日 平成30年2月15日(2018.2.15)
優先権データ
  • 特願2016-157484 (2016.8.10) JP
発明の名称 (英語) METHOD FOR PRODUCING GENE KNOCK-IN CELLS
発明の概要(英語) The present inventor attempted to produce a knock-in animal with the use of a single-stranded DNA as a donor by utilizing a genome editing system comprising a nuclease in the form of a protein and a DNA-targeting RNA. As a result, it was found that cells wherein the donor DNA was knocked-in could be thus obtained at an extremely high efficiency.
従来技術、競合技術の概要(英語) BACKGROUND ART
Gene targeting (knock-out or knock-in) is a mammal, in vivo analysis of gene function in an important tool that is, the embryonic stem cells (ES cells) can be produced by utilizing complex and time-consuming step and are therefore required.
According to this technique has been developed such as genome editing, ZFN,TALEN,CRISPR-Cas9, as a useful tool for modification of attention.
Genome editing of these techniques, currently the most used CRISPR-Cas9 system, immune-based mechanism of bacteria, an enzyme cutting double-stranded DNA and protein Cas9, a target DNA having a nucleotide sequence complementary to the region between the RNA (crRNA), and having a base sequence partially complementary crRNA RNA(trans-activationg RNA; tracrRNA) complex is made, to specifically recognize a target DNA region, and coupling, is cut.
Using this system, RNA encoding a protein Cas9, as well as and crRNA tracrRNA (these 2 one of the RNA is linker bonded via a single-stranded chimeric RNA including a case of the embodiment) is introduced into a fertilized egg, fertilized eggs in vivo directly operating in the genome (genome modified in vivo), without passing through ES cells, gene targeting can be manufactured a mammal (Patent Document 1, Non-Patent Document 1). To date, this approach allows the production of knockout mice (Patent Document 2, Non-Patent Document 2-4) and, with a single base substitution in the manufacture of the (non-patent document 3, 5, 6) knock-in mice has been performed a large number.
However, a relatively large system CRISPR-Cas9 of the gene was cloned into a mammalian case of manufacturing a knock, knock of the gene to be of very low efficiency problem (Non-Patent Document 7).
Therefore, the protein Cas9 (instead of RNA, in the form of proteins), crRNA fragments, and fragments of cloning free tracrRNA CRISPR-Cas9 system may be utilized, a relatively large size of the double-stranded DNA as a donor, a knock at high efficiency to a non-human mammal method has been developed (Patent Document 3). In this method, mainly, the donor is heterozygous DNA mammal a knock can be manufactured (see Comparative Example 2 described later).
On the other hand, using conventional CRISPR-Cas9 system, single-stranded DNA as a donor, a knock at high efficiency to a non-human mammal is also a method has been developed (Non-Patent Document 8). In this method, the donor is homozygous DNA to mammalian a knock can be successful.
Incidentally, in recent years, as a technique for a new genome editing, the system has been developed CRISPR-cpf1 (non-patent document 9). This system is also, in the same manner as system CRISPR-Cas9, and a guide RNA nuclease interacting with the genome editing techniques are used, the use of the system similar to the expected CRISPR-Cas9 are.
  • 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
  • NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL UNIVERSITY
  • 発明者(英語)
  • AIDA Tomomi
  • TANAKA Kohichi
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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