WO2014186686A3 - Targeted mutagenesis and genome engineering in plants using rna-guided cas nucleases - Google Patents

Targeted mutagenesis and genome engineering in plants using rna-guided cas nucleases Download PDF

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Publication number
WO2014186686A3
WO2014186686A3 PCT/US2014/038359 US2014038359W WO2014186686A3 WO 2014186686 A3 WO2014186686 A3 WO 2014186686A3 US 2014038359 W US2014038359 W US 2014038359W WO 2014186686 A3 WO2014186686 A3 WO 2014186686A3
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WO
WIPO (PCT)
Prior art keywords
plants
nucleic acid
methods
target nucleic
sgrna
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PCT/US2014/038359
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French (fr)
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WO2014186686A2 (en
Inventor
Jonathan D.G. Jones
Sophien KAMOUN
Vladimir NEKRASOV
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Two Blades Foundation
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Publication of WO2014186686A2 publication Critical patent/WO2014186686A2/en
Publication of WO2014186686A3 publication Critical patent/WO2014186686A3/en

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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
    • C12N15/63—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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells

Abstract

Methods for modifying the genome of plants at a target nucleic acid sequence are provided. Such methods involve producing a plant cell comprising a Cas protein and a single guide RNA (sgRNA) that is capable of recognizing the target nucleic acid sequence in the plant cell. Further provided are methods for targeting fusion proteins to target nucleic acid sequences in the genome of plant. Such methods involve producing a plant cell comprising a fusion protein and an sgRNA designed to recognize the target nucleic acid sequence. The fusion protein is capable of recognizing the target sequence in the presence of the sgRNA. The fusion protein comprises a Cas protein domain that is defective in nuclease activity and an additional domain. Also provided are methods for testing components of the Cas system in plants, modified plants and plant cells, fusion proteins, and nucleic acid molecules encoding such fusion proteins.
PCT/US2014/038359 2013-05-17 2014-05-16 Targeted mutagenesis and genome engineering in plants using rna-guided cas nucleases WO2014186686A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361824748P 2013-05-17 2013-05-17
US61/824,748 2013-05-17

Publications (2)

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WO2014186686A2 WO2014186686A2 (en) 2014-11-20
WO2014186686A3 true WO2014186686A3 (en) 2015-01-08

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WO2015133554A1 (en) 2014-03-05 2015-09-11 国立大学法人神戸大学 Genomic sequence modification method for specifically converting nucleic acid bases of targeted dna sequence, and molecular complex for use in same
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US11584936B2 (en) 2014-06-12 2023-02-21 King Abdullah University Of Science And Technology Targeted viral-mediated plant genome editing using CRISPR /Cas9
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CN107043779B (en) * 2016-12-01 2020-05-12 中国农业科学院作物科学研究所 Application of CRISPR/nCas 9-mediated site-specific base substitution in plants
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US20180258418A1 (en) * 2017-01-17 2018-09-13 Institute For Basic Science Method of identifying genome-wide off-target sites of base editors by detecting single strand breaks in genomic dna
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105131097A (en) * 2015-08-31 2015-12-09 中国林业科学研究院林业研究所 CabuAG gene related to pistil and stamen development of catalpa bungei and protein and application thereof
CN105131097B (en) * 2015-08-31 2018-10-09 中国林业科学研究院林业研究所 Chinese catalpa Pistil And Stamen develops relevant CabuAG genes and its protein and application
CN107937427A (en) * 2017-10-20 2018-04-20 广东石油化工学院 A kind of homologous repair vector construction method based on CRISPR/Cas9 systems
CN110331146A (en) * 2019-09-05 2019-10-15 中国科学院天津工业生物技术研究所 It is a kind of regulation sgRNA transcription promoter, expression vector and its genome editing system and application

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