WO2023112886A1 - Procédé de production d'arn monocaténaire - Google Patents

Procédé de production d'arn monocaténaire Download PDF

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Publication number
WO2023112886A1
WO2023112886A1 PCT/JP2022/045637 JP2022045637W WO2023112886A1 WO 2023112886 A1 WO2023112886 A1 WO 2023112886A1 JP 2022045637 W JP2022045637 W JP 2022045637W WO 2023112886 A1 WO2023112886 A1 WO 2023112886A1
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rna
protein
crispr
sequence
formula
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PCT/JP2022/045637
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English (en)
Japanese (ja)
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力也 渡邉
肇 篠田
弘志 西増
潤一郎 石川
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国立研究開発法人理化学研究所
国立大学法人東京大学
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Application filed by 国立研究開発法人理化学研究所, 国立大学法人東京大学 filed Critical 国立研究開発法人理化学研究所
Publication of WO2023112886A1 publication Critical patent/WO2023112886A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to a method for producing single-stranded RNA. More particularly, the present invention relates to methods for producing single-stranded RNA and populations of single-stranded RNA.
  • This application claims priority based on Japanese Patent Application No. 2021-204628 filed in Japan on December 16, 2021, the content of which is incorporated herein.
  • By-products include single-stranded RNA shorter than the single-stranded RNA of interest, single-stranded RNA complementary to the single-stranded RNA of interest, double-stranded RNA shorter than the single-stranded RNA of interest, and single-stranded RNA of interest. Examples include single-stranded RNA and double-stranded RNA in which a single-stranded RNA complementary to the single-stranded RNA of interest is hybridized.
  • One of the causes of by-products is known to be that the end of the synthesized single-stranded RNA serves as a primer to synthesize a complementary strand using itself as a template (self-primed extension).
  • Mu X., et al. An origin of the immunogenicity of in vitro transcribed RNA, Nucleic Acids Research, 46 (10), 5239-5249, 2018. Baiersdorfer M., et al., A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA, Molecular Therapy: Nucleic Acids Vol. 15, 26-35, 2019.
  • Gholamalipour Y., et al. Efficient inhibition of RNA self-primed extension by addition of competing 3'-capture DNA-improved RNA synthesis by T7 RNA polymerase, Nucleic Acids Research, 47 (19), e118, 2019. Shinoda H., et al., Amplification-free RNA detection with CRISPR-Cas13, Commun Biol., 4, 476, 2021.
  • An object of the present invention is to provide a technique for producing single-stranded RNA with a low contamination rate of by-products.
  • a method for producing a single-stranded RNA comprising the step of transcribing a DNA encoding an RNA represented by the following formula (1) to obtain an RNA mixture containing the single-stranded RNA to be produced and by-products; CRISPR-Cas12a protein or CRISPR-Cas13a protein is contacted with the RNA mixture, and a step of cleaving the 5' side or 3' side of the first base from the 5' end of R 1 in the following formula (1), a step of purifying the RNA mixture after cleaving the 5' side or 3' side of the first base from the 5' end of R1 in formula (1) to obtain a single-stranded RNA to be produced.
  • the production method, wherein the single-stranded RNA to be produced is a single-stranded RNA represented by the following formula (2) or (3).
  • T 1 represents an arbitrary RNA sequence
  • R 1 represents an RNA sequence recognized by CRISPR-Cas12a protein or CRISPR-Cas13a protein
  • T 2 is absent or an arbitrary Represents an RNA sequence
  • T 1 ' represents the same RNA sequence as T 1
  • R 1 ′ represents the same RNA sequence as R 1 or an RNA sequence obtained by adding the first base from the 5' end of the RNA sequence of R 1 to the 3' end of T 1
  • R 1 ′ represents the same RNA sequence as R 1 or an RNA sequence in which the first base from the 5′ end of the RNA sequence of R 1 is deleted.
  • T 1 represents an arbitrary RNA sequence
  • R 1 represents an RNA sequence recognized by CRISPR-Cas12a protein or CRISPR-Cas13a protein
  • T 2 is absent or an arbitrary Represents an RNA sequence
  • T 1 ' represents the same RNA sequence as T 1
  • R 1 ′ represents the same RNA sequence as R 1 or an RNA sequence in which the first base from the 5′ end of the RNA sequence of R 1 is deleted.
  • the by-product may comprise a partial fragment of RNA having a base sequence complementary to 5'-T 1 -R 1 -T 2 -3', e.g. It may contain RNA having a nucleotide sequence, may contain RNA having a nucleotide sequence complementary to 5'-R 1 -3', and is complementary to 5'-R 1 -T 2 -3' It may contain an RNA having a base sequence similar to the base sequence, or may contain an RNA having a base sequence complementary to 5′-T 2 -3′.
  • the RNA mixture may be contacted with the CRISPR-Cas12a protein.
  • CRISPR-Cas12a protein includes Cas12a protein orthologs, Cas12a protein variants, and the like.
  • Examples of the Cas12a protein that can be used in the production method of the present embodiment include, for example, Cas12a protein derived from Lachnospiraceae bacterium ND2006 (LbCas12a, UniProtKB accession number: A0A182DWE3), Acidaminococcus sp.
  • Cas12a protein (AsCas12a, UniProtKB accession number: U2UMQ6) from Francisella tularensis subsp.
  • 5'-aauuucuacuaaguguagau-3' (SEQ ID NO: 1) can be used as the base sequence of R1 .
  • AsCas12a is used as the Cas12a protein
  • 5'-aauuucuacucuuguagau-3' (SEQ ID NO: 2) can be used as the base sequence of R1 .
  • FnCas12a as the Cas12a protein
  • 5'-aauuucuacuguuguagau-3' SEQ ID NO: 3
  • T1 and T2 may be the same RNA sequence or may be different RNA sequences.
  • RNA with a length of 200 bases or more It is practically impossible to produce RNA with a length of 200 bases or more by solid-phase synthesis.
  • a population of single-stranded RNAs having a length of 200 bases or more and containing 0.1 mol% or less of RNA having a base sequence complementary to T2 . can be manufactured.
  • the content of RNA having a nucleotide sequence complementary to T2 can be measured, for example, by the SATORI method.
  • T 1 represents an arbitrary RNA sequence
  • R 1 represents an RNA sequence recognized by LwaCas13a or LtrCas13a
  • T 2 is absent or represents an arbitrary RNA sequence
  • T 1 ' represents an RNA sequence obtained by adding the first base from the 5' end of the RNA sequence of R1 to the 3' end of T1
  • R1 ' represents the first base from the 5' end of the RNA sequence of R1. base is deleted RNA sequence.
  • each cut gel was transferred to a new 1.5 mL tube.
  • 400 ⁇ L of RNase-free water was added to the excised gel, lightly vortexed, and incubated overnight at 4°C.
  • RNA was then concentrated by ethanol precipitation and dissolved in 50 ⁇ L of RNase-free water.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Procédé de production d'un ARN simple brin, ledit procédé comprenant : une étape de transcription d'un ADN codant pour un ARN de formule (1) pour obtenir un mélange contenant l'ARN simple brin et un sous-produit ; une étape de mise en contact du mélange avec Cas12a ou 13a pour cliver la partie 5'- ou 3'- de la base à l'extrémité 5' de R1 ; et une étape de purification du mélange pour obtenir l'ARN simple brin, l'ARN simple brin étant de formule (2) ou (3) (dans les formules : T1 et T2 représentent une séquence d'ARN arbitraire ; R1 représente la séquence de reconnaissance de Cas12a ou 13a ; T1' est identique à T1 ou représente une séquence dans laquelle la base à l'extrémité 5'- de R1 est ajoutée à l'extrémité 3'- de T1 ; et R1' est identique à R1 ou représente une séquence dans laquelle la base à l'extrémité 5'de R1 est supprimée).　(1) : 5'-T1-R1-T2-3' ; (2): 5'-T1-3' ; (3) : 5'-R1-T2-3'
PCT/JP2022/045637 2021-12-16 2022-12-12 Procédé de production d'arn monocaténaire WO2023112886A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021-204628 2021-12-16
JP2021204628 2021-12-16

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WO2023112886A1 true WO2023112886A1 (fr) 2023-06-22

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018111967A1 (fr) * 2016-12-13 2018-06-21 Modernatx, Inc. Purification par affinité d'arn
JP2018532402A (ja) * 2015-09-24 2018-11-08 クリスパー セラピューティクス アーゲー Ｒｎａプログラム可能エンドヌクレアーゼの新規のファミリーならびにゲノム編集および他の適用におけるそれらの使用
JP2019522472A (ja) * 2016-06-16 2019-08-15 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 標的ｒｎａを検出するための方法及び組成物
JP2019533476A (ja) * 2016-09-14 2019-11-21 モデルナティーエックス， インコーポレイテッド 高純度ｒｎａ組成物及びその調製のための方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018532402A (ja) * 2015-09-24 2018-11-08 クリスパー セラピューティクス アーゲー Ｒｎａプログラム可能エンドヌクレアーゼの新規のファミリーならびにゲノム編集および他の適用におけるそれらの使用
JP2019522472A (ja) * 2016-06-16 2019-08-15 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 標的ｒｎａを検出するための方法及び組成物
JP2019533476A (ja) * 2016-09-14 2019-11-21 モデルナティーエックス， インコーポレイテッド 高純度ｒｎａ組成物及びその調製のための方法
WO2018111967A1 (fr) * 2016-12-13 2018-06-21 Modernatx, Inc. Purification par affinité d'arn

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ALEXANDRA EAST-SELETSKY, MITCHELL R. O’CONNELL, SPENCER C. KNIGHT, DAVID BURSTEIN, JAMIE H. D. CATE, ROBERT TJIAN, JENNIFER : "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 538, no. 7624, 1 October 2016 (2016-10-01), London, pages 270 - 273, XP055719305, ISSN: 0028-0836, DOI: 10.1038/nature19802 *
INES FONFARA, RICHTER HAGEN, BRATOVIČ MAJDA, LE RHUN ANAÏS, CHARPENTIER EMMANUELLE: "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 532, no. 7600, 2016, London, pages 517 - 521, XP055349049, ISSN: 0028-0836, DOI: 10.1038/nature17945 *

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