WO2019234132A1 - Édition de bases dans des plantes déficientes en polymérase thêta - Google Patents

Édition de bases dans des plantes déficientes en polymérase thêta Download PDF

Info

Publication number
WO2019234132A1
WO2019234132A1 PCT/EP2019/064734 EP2019064734W WO2019234132A1 WO 2019234132 A1 WO2019234132 A1 WO 2019234132A1 EP 2019064734 W EP2019064734 W EP 2019064734W WO 2019234132 A1 WO2019234132 A1 WO 2019234132A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
nucleic acid
seq
acid sequence
cellular system
Prior art date
Application number
PCT/EP2019/064734
Other languages
English (en)
Inventor
Erik Jongedijk
Aaron HUMMEL
Derek Graham BARTLEM
Yu Mei
Original Assignee
KWS SAAT SE & Co. KGaA
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 KWS SAAT SE & Co. KGaA filed Critical KWS SAAT SE & Co. KGaA
Publication of WO2019234132A1 publication Critical patent/WO2019234132A1/fr

Links

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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • one of the one or more further DNA repair enzyme(s) of a NHEJ pathway is Ku70, or a nucleic acid sequence encoding the same, wherein the Ku70 comprises an amino acid sequence according to SEQ ID NO: 7- 9, or an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 7-9, respectively, or wherein the nucleic acid sequence encoding the same comprises a sequence according to SEQ ID NO: 10-12, or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 87%, 8
  • nucleic acid molecule or“nucleic acid sequence” used herein refer to single- or double-stranded DNA or RNA of natural or synthetic origin.
  • a nucleic acid molecule or a nucleic acid sequence comprises at least one nucleotide or two or more nucleotides, respectively, in a specific sequence of any length including oligonucleotides or polynucleotides.
  • the cellular system comprises the target ge- nome or genomic sequence to be modified in a suitable way, i.e., in a form accessible to a genetic modification or manipulation.
  • the cellular system may be selected from, for example, a prokaryotic or eukaryotic cell, including an animal or a plant cell, or the cellular system may comprise a genetic construct comprising all or parts of the genome of a prokaryotic or eukary- otic cell to be modified in a highly targeted way.
  • the cellular system may be provided as iso- lated cell or vector, or the cellular system may be comprised by a network of cells in a tissue, organ, material or whole organism, either in vivo or as isolated system in vitro.
  • the present invention provides a method to obtain transgene-free gene edited plants by deliv- ering base editing reagents into Polymerase theta-deficient cells or pol 0/lig4, pol 0/ku7O, pol 0/ku8O-double deficient cells.
  • the base editing reagents can be expressed transiently, regard- less of the delivery method (e.g., Agrobactium-mediated T-DNA delivery, biolistic delivery of circular plasmid or linearized DNA fragment) and amount of DNA delivered.
  • the delivery method e.g., Agrobactium-mediated T-DNA delivery, biolistic delivery of circular plasmid or linearized DNA fragment
  • RNA interference refers to a gene down-regulation mechanism mean- while demonstrated to exist in all eukaryotes. The mechanism was first recognized in plants where it was called “post-transcriptional gene silencing” or "PTGS".
  • RNAi small RNAs (of about 21-24 nucleotides) function to guide specific effector proteins (e.g., members of the Argonaute protein family) to a target nucleotide sequence by complementary base pairing. The effector protein complex then down-regulates the expression of the targeted RNA or DNA.
  • Small RNA-directed gene regulation systems were independently discovered (and named) in plants, fungi, worms, flies, and mammalian cells.
  • nucleic acid sequence encoding the same comprises a sequence according to SEQ ID NO: 10-12, or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%,
  • the predeter- mined location where the at least one base edit is induced is an endogene or a transgene in an organism of interest, wherein the endogene or the transgene is selected from the group consisting of a gene encoding resistance or tolerance to abiotic stress, including drought stress, osmotic stress, heat stress, cold stress, oxidative stress, heavy metal stress, nitrogen deficiency, phosphate deficiency, salt stress or waterlogging, herbicide resistance, including resistance to glyphosate, glufosinate/phosphinotricin, hygromycin, protoporphyrinogen oxi- dase (PPO) inhibitors, ALS inhibitors, and Dicamba, a gene encoding resistance or tolerance to biotic stress, including a viral resistance gene, a fungal resistance gene, a bacterial re- sistance gene, an insect resistance gene, or a gene encoding a yield related trait, including lodging resistance, flowering time,
  • the base edit induced in a regulatory sequence may result in an altered expression of one or more target gene(s).
  • an edited promoter sequence may show increased pro- moter activity, increased promoter tissue specificity, decreased promoter activity or decreased promoter tissue specificity compared to the unedited promoter sequence.
  • a new promoter activity, an inducible promoter activity, an extended window of gene expression, a modification of the timing or developmental progress of gene expression in the same cell layer or other cell layer, for example, extending the timing of gene expression in the tapetum of anthers, a mutation of DNA binding elements and/or a deletion or addition of DNA binding elements may result from the editing.
  • a fusion can for example provide for subcellular localization of the site-specific base editor (e.g., a nuclear localization signal (NLS) for targeting (e.g., a site-specific nuclease) to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast lo- calization signal for targeting to a chloroplast and the like.
  • a nuclear localization signal e.g., a site-specific nuclease
  • a mitochondrial localization signal for targeting to the mitochondria
  • chloroplast lo- calization signal for targeting to a chloroplast and the like.
  • the cellular system may comprise an inactivated or partially inactivated Polymerase theta enzyme and preferably one or more further inactivated or partially inacti- vated DNA repair enzyme(s) of a NHEJ pathway, and the cellular system comprises a modifi- cation as defined in any of the above aspects and embodiments, wherein the cellular system is selected from the group consisting of one or more plant cell(s), a plant, and parts of a plant.
  • wt Pol Q sequences genomic DNA - SEQ ID NO: 59, cDNA - SEQ ID NO: 60, protein - SEQ ID NO: 61
  • tobacco Nicotiana tabacum
  • wt Pol Q sequences genomic DNA - SEQ ID NO: 62, cDNA - SEQ ID NO: 63, protein - SEQ ID NO: 64
  • CRISPR/Cas technology please see also protocol below:“NT PolQ KO construct and KO event detection", Nature Plants 2:16164, 2016.
  • a transformation protocol for tobacco can be found in Fisher and Guiltinan, Plant Molecular Biology Reporter, 1995, vol. 13, issue 3, 278-289.
  • KO vector was constructed, which was named as pHUE41 1- POLQ-T. Rice transformation is performed with Agrobacterium-mediated method using Agro strain AGL1. The protocol can be found in the reference Hiei et al., The Plant Journal, 1994, 6(2), 271-282.
  • NtPol theta target site 1 ATTAACAGACTGCTAGAAGAGGG (PAM) (SEQ ID NO: 1 1 1 )
  • NtALS-12 Two target sgRNA were constructed in the same plasmid named as NtALS-12 ( Figure 15A).
  • the transformed tobacco tissue is regenerated on selection media containing a sulfonylurea (Fisher and Guiltinan, 1995, vide supra).
  • the herbicide resistant plant would have undergone a desired base conversion, which is further confirmed by sequencing analysis.
  • the pres- ence/absence of the transgenic DNA is also screened using the same method as in Example 2.
  • Figure 3 shows the genomic sequence of ALS from Nicotiana tabacum (NtALS, SEQ ID NO: 56).
  • the coding sequence is underlined, gRNA complementary region is in italic, including the PAM, which is in bold.
  • the desired C to T conversion site is shaded.
  • the first two target sgRNA were constructed in the same plasmid named as osPDS-12 and the other two were constructed in the same plasmid named as osPDS-34 ( Figure 16).
  • the derived rice plants are screened through a similar process as in Example 2.
  • Figure 4 shows the genomic sequence of CDS from Oryza sativa (OsPDS, SEQ ID NO: 57).
  • the coding se- quence is underlined, the gRNA complementary region is in italic, including the PAM, which is in bold.
  • the desired C to T conversion sites are shaded.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention concerne des procédés améliorés pour l'édition à un site spécifique d'un ou plusieurs nucléotides dans un matériel génétique d'un système cellulaire, moyennant quoi l'intégration aléatoire de séquences dans le matériel génétique est significativement réduite ou complètement évitée, la polymérase têta du système cellulaire étant inactivée ou partiellement inactivée et, de préférence, une ou plusieurs autres enzymes d'une voie NHEJ étant également inactivées ou partiellement inactivées. L'invention concerne en outre des systèmes cellulaires obtenus par le procédé selon l'invention et en particulier des plantes ou des parties de plantes, qui comprennent une ou plusieurs éditions de bases spécifiques mais ne comprennent pas de séquences transgéniques.
PCT/EP2019/064734 2018-06-05 2019-06-05 Édition de bases dans des plantes déficientes en polymérase thêta WO2019234132A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US201862680867P 2018-06-05 2018-06-05
US62/680,867 2018-06-05
US201862711747P 2018-07-30 2018-07-30
US62/711,747 2018-07-30
US201862731434P 2018-09-14 2018-09-14
US62/731,434 2018-09-14

Publications (1)

Publication Number Publication Date
WO2019234132A1 true WO2019234132A1 (fr) 2019-12-12

Family

ID=66998345

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2019/064734 WO2019234132A1 (fr) 2018-06-05 2019-06-05 Édition de bases dans des plantes déficientes en polymérase thêta

Country Status (1)

Country Link
WO (1) WO2019234132A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112522302A (zh) * 2020-12-11 2021-03-19 安徽省农业科学院水稻研究所 水稻双向单碱基编辑的共转录单元基因abe-cbe系统及其应用
WO2022090224A1 (fr) * 2020-10-27 2022-05-05 KWS SAAT SE & Co. KGaA Utilisation d'une activité pol thêta améliorée pour l'ingénierie génomique eucaryote

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015089406A1 (fr) 2013-12-12 2015-06-18 President And Fellows Of Harvard College Variantes genetiques de cas pour l'edition genique
WO2015133554A1 (fr) 2014-03-05 2015-09-11 国立大学法人神戸大学 Procédé de modification de séquence génomique permettant la conversion de façon spécifique de bases d'acide nucléique de séquences d'adn ciblées et complexe moléculaire destiné à être utilisée dans ce dernier
US20150307889A1 (en) * 2014-04-28 2015-10-29 Dow Agrosciences Llc Haploid maize transformation
WO2016030019A1 (fr) * 2014-08-28 2016-03-03 Kws Saat Se Production de plantes haploïdes
WO2017004375A1 (fr) * 2015-06-30 2017-01-05 Regents Of The University Of Minnesota Lignée inductrice d'haploïdes pour l'édition de génome accélérée
WO2017062754A1 (fr) * 2015-10-07 2017-04-13 New York University Compositions et procédés pour augmenter l'activité crispr par inhibition de polq
EP3159413A1 (fr) * 2015-10-22 2017-04-26 Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK); OT Gatersleben Génération de plantes haploïdes basées sur knl2
WO2017070633A2 (fr) 2015-10-23 2017-04-27 President And Fellows Of Harvard College Protéines cas9 évoluées pour l'édition génétique
WO2017164738A1 (fr) * 2016-03-24 2017-09-28 Universiteit Leiden Procédés pour transfecter des plantes et réduire les événements d'intégration aléatoire
WO2018027078A1 (fr) 2016-08-03 2018-02-08 President And Fellows Of Harard College Éditeurs de nucléobases d'adénosine et utilisations associées
WO2019086460A1 (fr) * 2017-10-30 2019-05-09 Kws Saat Se Nouvelles stratégies d'édition génomique de précision

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015089406A1 (fr) 2013-12-12 2015-06-18 President And Fellows Of Harvard College Variantes genetiques de cas pour l'edition genique
WO2015133554A1 (fr) 2014-03-05 2015-09-11 国立大学法人神戸大学 Procédé de modification de séquence génomique permettant la conversion de façon spécifique de bases d'acide nucléique de séquences d'adn ciblées et complexe moléculaire destiné à être utilisée dans ce dernier
US20150307889A1 (en) * 2014-04-28 2015-10-29 Dow Agrosciences Llc Haploid maize transformation
WO2016030019A1 (fr) * 2014-08-28 2016-03-03 Kws Saat Se Production de plantes haploïdes
WO2017004375A1 (fr) * 2015-06-30 2017-01-05 Regents Of The University Of Minnesota Lignée inductrice d'haploïdes pour l'édition de génome accélérée
WO2017062754A1 (fr) * 2015-10-07 2017-04-13 New York University Compositions et procédés pour augmenter l'activité crispr par inhibition de polq
EP3159413A1 (fr) * 2015-10-22 2017-04-26 Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK); OT Gatersleben Génération de plantes haploïdes basées sur knl2
WO2017070633A2 (fr) 2015-10-23 2017-04-27 President And Fellows Of Harvard College Protéines cas9 évoluées pour l'édition génétique
WO2017070632A2 (fr) 2015-10-23 2017-04-27 President And Fellows Of Harvard College Éditeurs de nucléobases et leurs utilisations
WO2017164738A1 (fr) * 2016-03-24 2017-09-28 Universiteit Leiden Procédés pour transfecter des plantes et réduire les événements d'intégration aléatoire
WO2018027078A1 (fr) 2016-08-03 2018-02-08 President And Fellows Of Harard College Éditeurs de nucléobases d'adénosine et utilisations associées
WO2019086460A1 (fr) * 2017-10-30 2019-05-09 Kws Saat Se Nouvelles stratégies d'édition génomique de précision

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
FISHERGUILTINAN, PLANT MOLECULAR BIOLOGY REPORTER, vol. 13, no. 3, 1995, pages 278 - 289
GAUDELLI ET AL., NATURE, vol. 551, 2017, pages 464 - 471
HA J.S. ET AL., JOURNAL OF CONTROLLED RELEASE, vol. 250, 2017, pages 27 - 35
HIEI ET AL., THE PLANT JOURNAL, vol. 6, no. 2, 1994, pages 271 - 282
HUA ET AL., MOLECULAR PLANT, vol. 11, no. 4, 2018, pages 627 - 630
KLEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 8502 - 8505
KOMOR ET AL., NATURE, vol. 533, no. 7603, 2016, pages 420 - 424
MAARTJE VAN KREGTEN ET AL: "T-DNA integration in plants results from polymerase-[theta]-mediated DNA repair", NATURE PLANTS, vol. 2, no. 11, 31 October 2016 (2016-10-31), XP055552907, DOI: 10.1038/nplants.2016.164 *
NATURE PLANTS, vol. 2, 2016, pages 16164
PRASAD ET AL., NUCLEIC ACID RESEARCH, vol. 37, no. 6, 2009, pages 1868 - 1877
SMITH, T.F.WATERMAN, M.S.: "Identification of common molecular subsequences", JOURNAL OF MOLECULAR BIOLOGY, vol. 147, no. 1, 1981, pages 195 - 197, XP024015032, DOI: doi:10.1016/0022-2836(81)90087-5
VAN KREGTEN ET AL.: "NT PolQ KO construct and KO event detection", NATURE PLANTS, vol. 2, 2016, pages 16164
XING ET AL., BMC PLANT BIOLOGY, vol. 14, 2014, pages 327
XING ET AL., MBC PLANT BIOLOGY, vol. 14, 2014, pages 327
YOSHIMURA ET AL., MOLECULAR CELL, vol. 24, no. 1, 2006, pages 115 - 125
ZELENSKY ET AL., NATURE COMMUNICATIONS, vol. 8, 2017
ZONG ET AL., NATURE BIOTECHNOLOGY, vol. 25, no. 5, 2017, pages 438 - 440

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022090224A1 (fr) * 2020-10-27 2022-05-05 KWS SAAT SE & Co. KGaA Utilisation d'une activité pol thêta améliorée pour l'ingénierie génomique eucaryote
CN112522302A (zh) * 2020-12-11 2021-03-19 安徽省农业科学院水稻研究所 水稻双向单碱基编辑的共转录单元基因abe-cbe系统及其应用
CN112522302B (zh) * 2020-12-11 2022-04-29 安徽省农业科学院水稻研究所 水稻双向单碱基编辑的共转录单元基因abe-cbe系统及其应用

Similar Documents

Publication Publication Date Title
EP3382019B1 (fr) Procédé de conversion de séquence de génome de monocotylédone convertissant spécifiquement une base d'acide nucléique d'une séquence adn ciblée, et complexe moléculaire mettant en oeuvre celui-ci
EP3036332B1 (fr) Modification du génome des plantes à l'aide de systèmes d'arn de guidage/endonucléase cas et leurs procédés d'utilisation
Srivastava et al. Cre‐mediated site‐specific gene integration for consistent transgene expression in rice
CN110157726B (zh) 植物基因组定点替换的方法
US20210403901A1 (en) Targeted mutagenesis using base editors
EP0959133A1 (fr) Procédé pour l'inhibtion de l' expression de gènes
US11578334B2 (en) Targeted endonuclease activity of the RNA-guided endonuclease CasX in eukaryotes
US20210163968A1 (en) Optimized plant crispr/cpf1 systems
CN110709519A (zh) 表达调控元件及其用途
US20210348179A1 (en) Compositions and methods for regulating gene expression for targeted mutagenesis
US20210155948A1 (en) Method for increasing the expression level of a nucleic acid molecule of interest in a cell
EP4234701A2 (fr) Régénération de plantes génétiquement modifiées
CA3080864A1 (fr) Nouvelles strategies d'edition genomique de precision
WO2019234132A1 (fr) Édition de bases dans des plantes déficientes en polymérase thêta
CA2460617A1 (fr) Procede pour modifier le genome dans une plante superieure
KR20190122595A (ko) 식물의 염기 교정용 유전자 구조체, 이를 포함하는 벡터 및 이를 이용한 염기 교정 방법
JP7452884B2 (ja) Ｄｎａが編集された植物細胞を製造する方法、及びそれに用いるためのキット
US20220340919A1 (en) Promoter repression
WO2023111130A1 (fr) Agrobactéries modifiées pour modifier des plantes
CN116855532A (zh) 一种用于骨干载体的RNAi表达框及相应载体和应用
AU1157399A (en) Methods for obtaining plant varieties

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19731888

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19731888

Country of ref document: EP

Kind code of ref document: A1