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WO2025135134A1 - Complexe nucléase guide d'arn de type tnpb - Google Patents

Complexe nucléase guide d'arn de type tnpb Download PDF

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Publication number
WO2025135134A1
WO2025135134A1 PCT/JP2024/045047 JP2024045047W WO2025135134A1 WO 2025135134 A1 WO2025135134 A1 WO 2025135134A1 JP 2024045047 W JP2024045047 W JP 2024045047W WO 2025135134 A1 WO2025135134 A1 WO 2025135134A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
sequence
tnpb
guided nuclease
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2024/045047
Other languages
English (en)
Japanese (ja)
Inventor
彰良 中村
茂夫 菅野
宏 山本
展隆 光田
輝彦 寺川
翼 矢野
玲花 長谷川
誠一郎 伊藤
尚美 白井
佑里 二宮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Institute of Advanced Industrial Science and Technology AIST
Inplanta Innovations Inc
Toppan Holdings Inc
Original Assignee
National Institute of Advanced Industrial Science and Technology AIST
Inplanta Innovations Inc
Toppan Holdings Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Institute of Advanced Industrial Science and Technology AIST, Inplanta Innovations Inc, Toppan Holdings Inc filed Critical National Institute of Advanced Industrial Science and Technology AIST
Priority to JP2025555860A priority Critical patent/JPWO2025135134A1/ja
Publication of WO2025135134A1 publication Critical patent/WO2025135134A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • a mutant protein may be specified as having 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more homology and/or identity to the amino acid sequence of the original protein.
  • the mutant protein belongs to the same clade as the original protein.
  • the homology or identity is usually preferably 50% or more, 60% or more, 70% or more, or 80% or more.
  • Gene introduction can be performed using methods known in the art. There are no particular limitations on the introduction method, and it can be selected appropriately depending on the type of thing to be introduced and the target of introduction.
  • Introduction methods are broadly divided into direct methods and methods using viral vectors. Direct methods include electroporation, liposome methods, particle gun methods in which gold particles are injected together, and whisker methods.
  • direct methods include electroporation, liposome methods, particle gun methods in which gold particles are injected together, and whisker methods.
  • adenovirus, adeno-associated virus, lentivirus, Agrobacterium, tobacco mosaic virus (TMV), etc. can be used as vectors depending on the host species.
  • Target-specific cleavage in cells can be repaired by a genome DNA repair mechanism. Non-homologous end joining or homologous recombination occurs during this process, making it possible to knock out or knock in genes. Therefore, the target-specific cleavage method of the present invention can also be called a genome editing method.
  • the genome editing method of the present invention may be performed on cultured cells, cultured tissues, or living cells.
  • the target species is not particularly limited, but genome editing is possible for any cell of bacteria, archaea, or eukaryotes.
  • the eukaryote may be any cell, such as a plant cell, insect cell, or animal cell, and genome editing is possible for any mammalian cell, for example, and genome editing is possible for both human cells and non-human animal cells.
  • the T protein of the present invention is characterized by a low optimum temperature. Therefore, genome editing can be performed at a temperature of 10 to 30°C.
  • Another aspect of the present invention relates to a method for recruiting a specific protein to a target DNA using the TnpB-like RNA-guided nuclease complex of the present invention, which is engineered so as not to exert TnpB-like RNA-guided nuclease activity.
  • the activity of TnpB-like RNA-guided nuclease may be inactivated, or the length of the guide RNA may be adjusted to use a guide RNA that contains a sequence of a length that allows TnpB-like protein to bind specifically to a target while not exhibiting DNA cleavage activity.
  • a guide sequence of a target sequence of about 10 bases may be used.
  • the TnpB-like RNA-guided nuclease complex used in the method for recruiting a specific protein to a target DNA is as follows: (i) a TnpB-like RNA-guided nuclease according to the present invention in which an amino acid residue at the active center is replaced with another amino acid; (ii) a guide RNA consisting of a sequence including a guide sequence and a gRNA scaffold sequence; and (iii) a specific protein that binds to or interacts with the TnpB-like RNA-guided nuclease.
  • the complex By expressing or introducing such a complex into a cell, the complex is recruited near the site of the guide sequence, while no cleavage occurs, and a specific protein bound to the complex can be recruited.
  • An example of the active center of the mutant TnpB-like RNA-guided nuclease is a typical DED active center contained in the RuvC domain.
  • the activity of the TnpB-like guided nuclease can be inactivated by introducing mutations into D308, E489, and D575.
  • the mutation can be appropriately selected within a range that can inactivate the activity, and an example thereof is a mutation to alanine.
  • the RNA scaffold sequence of the guide RNA described in (ii) can be inserted with a binding site for other proteins such as the MS2 sequence.
  • the specific protein described in (iii) is a protein that modifies DNA or epigenetic states, specifically, all proteins commonly referred to as epigenetic factors are available. More specifically, p300, DMNT3, KRAB, SRDX, VPR, VP64, etc.
  • epigenetic factors are available. More specifically, p300, DMNT3, KRAB, SRDX, VPR, VP64, etc.
  • light manipulation tools such as fluorescent proteins (specifically, APOBEC, AID, TadA, Dda, FokI, M-MLV-RTase, GFP, etc.) are performed, but are not limited thereto.
  • the epigenetic state can be changed in the target DNA region.
  • kits Another aspect of the present invention may relate to a kit for cleaving a target nucleic acid in a cell or a kit for genome editing.
  • an expression cassette for the TnpB-like RNA-guided nuclease of the present invention (ii) An expression cassette of a guide RNA having a sequence including a gRNA scaffold sequence.
  • the expression cassette of the guide RNA is provided so that a guide sequence can be inserted.
  • the expression cassette of the guide RNA can be prepared by designing a cleavage target sequence and introducing a guide sequence.
  • the expression cassette of the TnpB-like RNA guide nuclease and the expression cassette of the guide RNA into which the guide sequence has been introduced are incorporated into a plasmid or vector, for example, and then genetically introduced into a cell.
  • An expression cassette usually contains a promoter sequence, and can be created by arranging a sequence encoding a TnpB-like RNA-guided nuclease or a guide RNA under its control.
  • the promoter can control the expression of a downstream sequence either transiently or constantly.
  • the expression cassette may be contained on one polynucleotide or on different polynucleotides.
  • RNA Clean & Concentrator-5 spin column Zymo Research
  • 13.6 units of DNAse QIAGEN
  • the bound RNA was eluted with 15 ⁇ l of nuclease-free water.
  • 500 ng of purified RNA was then used for RNA library preparation.
  • RNA libraries were prepared using SMARTer smRNA-Seq Kit for Illumina (TAKARA-BIO) according to the manufacturer's instructions.
  • the resulting NGS adaptor-ligated cDNA was amplified by PCR for eight cycles using full-length Illumina NGS indexing primers.
  • HEK293FT cells After culturing for 3 days, the DNA of HEK293FT cells was extracted with an alkaline buffer (0.1 N NaOH) and roughly purified at 100 pg/ ⁇ L as a template.
  • KOD One PCR master Mix and a first round PCR primer set sequence numbers 110 and 111 when any of gRNA1, gRNA2, and gRNA3 was used, and sequence numbers 112 and 113 when any of gRNA4, gRNA5, and gRNA6 was used
  • a partial sequence of the hAAVS1 gene containing the genome editing target sequence was subjected to a PCR reaction under the following conditions to amplify the target sequence.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Le but de la présente invention est de fournir : une nouvelle nucléase de guidage d'ARN qui peut être appliquée à une technique d'édition de génome ; et son utilisation. L'invention concerne une nucléase guide d'ARN de type TnpB qui fonctionne dans un complexe endonucléase de guidage d'ARN qui peut être appliqué à une technique d'édition génomique. En conséquence, l'invention concerne un complexe de nucléase guide d'ARN de type TnpB comprenant une nucléase guide d'ARN de type TnpB et un ARNg ayant une séquence de guidage et une séquence d'échafaudage d'ARNg.
PCT/JP2024/045047 2023-12-19 2024-12-19 Complexe nucléase guide d'arn de type tnpb Pending WO2025135134A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2025555860A JPWO2025135134A1 (fr) 2023-12-19 2024-12-19

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2023-214275 2023-12-19
JP2023214275 2023-12-19

Publications (1)

Publication Number Publication Date
WO2025135134A1 true WO2025135134A1 (fr) 2025-06-26

Family

ID=96137170

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2024/045047 Pending WO2025135134A1 (fr) 2023-12-19 2024-12-19 Complexe nucléase guide d'arn de type tnpb

Country Status (2)

Country Link
JP (1) JPWO2025135134A1 (fr)
WO (1) WO2025135134A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022159892A1 (fr) * 2021-01-25 2022-07-28 The Broad Institute, Inc. Polypeptides tnpb reprogrammables et leur utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022159892A1 (fr) * 2021-01-25 2022-07-28 The Broad Institute, Inc. Polypeptides tnpb reprogrammables et leur utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALTAE-TRAN, HAN : "Diversity, evolution, and classification of the RNA-guided nucleases TnpB and Cas12. ", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 120, no. 48, 1 January 2023 (2023-01-01), XP093325008, DOI: 10.1073/pnas.2308224120 *
DATABASE Protein 28 August 2021 (2021-08-28), ANONYMOUS: "transposase [Rhizobium laguerreae] ", XP093324984, Database accession no. WP_222046788.1 *
DATABASE WP_259185500.1 2 September 2022 (2022-09-02), ANONYMOUS: "transposase [Rhizobium sp. BK176] ", XP093324997, Database accession no. Protein *
NAKAGAWA RYOYA, HIRANO HISATO, OMURA SATOSHI N., NETY SUCHITA, KANNAN SOUMYA, ALTAE-TRAN HAN, YAO XIAO, SAKAGUCHI YURIKO, OHIRA TA: "Cryo-EM structure of the transposon-associated TnpB enzyme", NATURE, SPRINGER NATURE LIMITED, LONDON, vol. 616, no. 7956, 13 April 2023 (2023-04-13), London, pages 390 - 397, XP093272444, ISSN: 0028-0836, DOI: 10.1038/s41586-023-05933-9 *

Also Published As

Publication number Publication date
JPWO2025135134A1 (fr) 2025-06-26

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