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Artificial single guide RNA and use thereof

An independent and atomic technology, applied in the field of artificial single guide RNA, can solve the problems of culture, enzyme reaction time and labor, and natural RNA instability

Active Publication Date: 2018-12-21
BONAC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] As mentioned above, in genome editing using the CRISPR / Cas9 system, introducing sgRNA in the form of RNA has certain advantages, but on the contrary, there are the following problems: natural RNA is unstable in vivo, and recombination, based on in vitro When manufacturing by transcription, preparation of vector and template DNA is required, and time and labor are required for cultivation, enzyme reaction, and RNA purification

Method used

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  • Artificial single guide RNA and use thereof
  • Artificial single guide RNA and use thereof
  • Artificial single guide RNA and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0351] (Example 1) Synthesis of sgRNA

[0352] The sgRNA shown in Table 1 was synthesized based on the phosphoramidite method and using a nucleic acid synthesizer (The ABI Expedite(R) 8909 Nucleic Acid Synthesis System, Applied Biosystems). The aforementioned synthesis used EMM imide as RNA imide (International Publication No. 2013 / 027843) (the same applies below). The aforementioned imide was deprotected according to a prescribed method, and purified by HPLC. In the following sequence, the underline is the guide region complementary to the target nucleotide sequence, proline diamide imide is introduced into P, lysine diamide imide is introduced into K, and glycylglycine is introduced into X Diamide imide.

[0353] [Table 1]

[0354]

Embodiment 2

[0371] (Embodiment 2) Phenylalanine imide: the synthesis of compound (5)

[0372] Compound (5) was synthesized according to the following scheme.

[0373]

[0374] [[1] Synthesis of compound (1)]

[0375] In Fmoc-phenylalanine (3.0g, 7.7mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (1.78g, 9.3mmol) 1-Hydroxybenzotriazole monohydrate (HOBt) (2.51g, 18.6mmol) in acetonitrile solution (100mL) was added 4-amino-1-butanol (0.83g, 9.3mmol) and stirred overnight at room temperature . After completion of the reaction, the solvent was distilled off under reduced pressure. Dichloromethane was added to the residue, and the residue was washed twice with saturated aqueous sodium bicarbonate solution and once with saturated aqueous sodium chloride solution. The washed organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product (4.1 g) of the target compound (1).

[0376] [[2] Synthesis of compou...

Embodiment 3

[0391] (Embodiment 3) Leucine imide: the synthesis of compound (10)

[0392] Compound (10) was synthesized according to the following scheme.

[0393]

[0394] [[1] Synthesis of compound (6)]

[0395] In Fmoc-leucine (3.0g, 8.5mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (1.95g, 10.2mmol), 1-Hydroxybenzotriazole monohydrate (HOBt) (2.75 g, 20.4 mmol) in acetonitrile (90 mL) was added with 4-amino-1-butanol (0.91 g, 10.2 mmol) and stirred overnight at room temperature. After completion of the reaction, the solvent was distilled off under reduced pressure. Dichloromethane was added to the residue, and the residue was washed twice with saturated aqueous sodium bicarbonate solution and once with saturated aqueous sodium chloride solution. The washed organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product (4.42 g) of the target compound (6).

[0396] [[2] Synthesis of compound (7)] ...

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Abstract

Provided are: a novel artificial single guide RNA (sgRNA) that has an activity equal to or higher than the activity of a natural type sgRNA and shows an improved in vivo stability; and an efficient CRISPR / Cas9 system comprising a combination of the artificial sgRNA with a Cas9. When a nucleotide linker moiety for single-strand formation, said nucleotide linker moiety linking the 3'-end of a crRNAto the 5'-end of a tracrRNA in the sgRNA, is substituted by an amino acid derivative linker, the resultant sgRNA still sustains an activity equal to or higher than the activity of the original sgRNA in an in vitro DNA cleavage assay. When a linker region disposed between stem-loop 1 and stem-loop 2 in the tracrRNA and / or the loop moiety of stem-loop 2 are substituted by an amino acid derivative linker, or when an amino acid derivative linker is added / inserted in the vicinity of the 5'-end and / or the 3'-end of the sgRNA, the resultant sgRNA still sustains an activity equal to or higher than theactivity of the original sgRNA. By introducing one or more amino acid derivative linkers into the sgRNA, the in vivo stability thereof can be improved.

Description

technical field [0001] The present invention relates to an artificial single guide RNA (hereinafter also referred to as "sgRNA") that has activity equal to or higher than that of the natural type and has improved stability in vivo, and a combination of the artificial sgRNA and CRISPR-related protein 9 ( CRISPR-associated protein 9: Cas9) CRISPR / Cas9 system, and uses thereof. Background technique [0002] Clustered regularly interspaced short palindromic repeats (CRISPR: clustered regularly interspacedshort palindromic repeats) are short repetitive sequences of tens of base pairs that exist in bacteria and are involved in the acquisition of foreign DNA for phages, plasmids, etc. immune mechanism. One of the Cas genomes upstream of CRISPR recognizes a specific short sequence called a proto-spacer adjacent motif (PAM: proto-spacer adjacent motif) in the foreign DNA, and cuts out tens of base pairs upstream of it , inserted into the CRISPR region. After the inserted target se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07C229/04C07C229/34C12N9/16C12N15/11
CPCC07C229/04C07C229/34C12N9/16C12N15/09C12N15/113C12N2310/3181C12N2320/51C12N2330/30C12N2310/20Y02P20/55C12N9/22C12N15/102C12N15/907C07F9/222C12N15/11C12N2800/80C40B50/06
Inventor 青木绘里子大木忠明木下高志
Owner BONAC CORP
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