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METHOD FOR PRODUCING GENE KNOCK-IN CELLS

Foreign code F180009356
File No. (S2016-0981-N0)
Posted date Apr 19, 2018
Country WIPO
International application number 2017JP027838
International publication number WO 2018030208
Date of international filing Aug 1, 2017
Date of international publication Feb 15, 2018
Priority data
  • P2016-157484 (Aug 10, 2016) JP
Title METHOD FOR PRODUCING GENE KNOCK-IN CELLS
Abstract 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.
Outline of related art and contending technology 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.
Scope of claims (In Japanese)[請求項1]
所望のDNAが標的DNA領域に挿入された細胞の作製方法であって、
(a)ヌクレアーゼ活性を有するタンパク質、
(b)標的DNA領域の塩基配列に対して相補的な塩基配列及び(a)のタンパク質と相互作用する塩基配列を含むDNA標的化RNA、及び
(c)所望のDNAを含む一本鎖ドナーDNA
を細胞に導入する工程を含む方法。
[請求項2]
所望のDNAが標的DNA領域に挿入された細胞の作製方法であって、
(a)Cas9タンパク質、
(b)crRNA断片とtracrRNA断片の組み合わせ、及び
(c)所望のDNAを含む一本鎖ドナーDNA
を細胞に導入する工程を含む方法。
[請求項3]
(c)の一本鎖ドナーDNAが600塩基以上の塩基配列を有する、請求項1又は2に記載の方法。
[請求項4]
(c)の一本鎖ドナーDNAが、次の(i)又は(ii)の方法で調製される、請求項1~3のいずれかに記載の方法。
(i)量の異なる一対のプライマーを用いたPCRによりDNAを増幅する工程、及び増幅産物に含まれる二本鎖DNAを特異的に分解する工程を含む方法
(ii)一対のプライマーのうち、一方のプライマーにリン酸化されたプライマーを用いるPCRによりDNAを増幅する工程、及び増幅産物におけるリン酸化された鎖を特異的に分解する工程を含む方法
[請求項5]
細胞が卵母細胞である、請求項1~4のいずれか1項に記載の方法。
[請求項6]
卵母細胞が受精卵である、請求項5に記載の方法。
[請求項7]
所望のDNAが標的DNA領域に挿入された細胞を含む非ヒト個体の作製方法であって、
(a)請求項1~6のいずれか1項に記載の方法を実施する工程、及び
(b)工程(a)により得られた細胞から非ヒト個体を作製する工程、
を含む方法。
[請求項8]
非ヒト哺乳動物がげっ歯類である、請求項7に記載の方法。
[請求項9]
請求項1~8のいずれか1項に記載の方法に用いるためのキットであって、以下の(a)から(c)からなる群より選択される少なくとも1の分子を含むキット。
(a)ヌクレアーゼ活性を有するタンパク質
(b)標的DNA領域の塩基配列に対して相補的な塩基配列及び(a)のタンパク質と相互作用する塩基配列を含むDNA標的化RNA
(c)所望のDNAを含む一本鎖ドナーDNA
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL UNIVERSITY
  • Inventor
  • AIDA Tomomi
  • TANAKA Kohichi
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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