Single and multiple gene knockouts by CRISPR–Cas9 in maize

Autor: Peter M. Rogowsky, Virginia Maria Grazia Borrelli, Jérémy Just, Laurine M. Gilles, Ghislaine Gendrot, Yannick Fierlej, Marie-France Gérentes, Nicolas M. Doll, Thomas Widiez, Christelle Richard, Gwyneth C. Ingram
Přispěvatelé: Reproduction et développement des plantes (RDP), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-École normale supérieure - Lyon (ENS Lyon), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Dauphine Recherches en Management (DRM), Université Paris Dauphine-PSL, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Investissement d'Avenir program of the French National Agency of Research for the project GENIUS ANR-11-BTBR-0001_GENIUS, INRA Plant Science and Breeding Division, CIFRE fellowships of the ANRT 2015/0777 2018/0480, Doctoral School on the Agro-Food System (Agrisystem) of Universita Cattolica del Sacro Cuore (Italy), Ministere de l'Enseignement Superieur et de la Recherche, ANR-11-BTBR-0001,GENIUS,Ingénierie cellulaire : amélioration et innovation technologiques pour les plantes d'une agriculture(2011), École normale supérieure - Lyon (ENS Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Dauphine-PSL, Unité de recherche en génomique végétale (URGV), Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Recherche Agronomique (INRA), École normale supérieure de Lyon (ENS de Lyon)-Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Institut National de la Recherche Agronomique (INRA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)
Jazyk: angličtina
Rok vydání: 2019
Předmět:
Zdroj: Plant Cell Reports
Plant Cell Reports, Springer Verlag, 2019, 38 (4), pp.487-501. ⟨10.1007/s00299-019-02378-1⟩
Plant Cell Reports, 2019, 38 (4), pp.487-501. ⟨10.1007/s00299-019-02378-1⟩
ISSN: 0721-7714
1432-203X
DOI: 10.1007/s00299-019-02378-1⟩
Popis: International audience; The analysis of 93 mutant alleles in 18 genes demonstrated that CRISPR-Cas9 is a robust tool for targeted mutagenesis in maize, permitting efficient generation of single and multiple knockouts. CRISPR-Cas9 technology is a simple and efficient tool for targeted mutagenesis of the genome. It has been implemented in many plant species, including crops such as maize. Here we report single- and multiple-gene mutagenesis via stably transformed maize plants. Two different CRISPR-Cas9 vectors were used allowing the expression of multiple guide RNAs and different strategies to knockout either independent or paralogous genes. A total of 12 plasmids, representing 28 different single guide RNAs (sgRNAs), were generated to target 20 genes. For 18 of these genes, at least one mutant allele was obtained, while two genes were recalcitrant to sequence editing. 19% (16/83) of mutant plants showed biallelic mutations. Small insertions or deletions of less than ten nucleotides were most frequently observed, regardless of whether the gene was targeted by one or more sgRNAs. Deletions of defined regions located between the target sites of two guide RNAs were also reported although the exact deletion size was variable. Double and triple mutants were created in a single step, which is especially valuable for functional analysis of genes with strong genetic linkage. Off-target effects were theoretically limited due to rigorous sgRNA design and random experimental checks at three potential off-target sites did not reveal any editing. Sanger chromatograms allowed to unambiguously class the primary transformants; the majority (85%) were fully edited plants transmitting systematically all detected mutations to the next generation, generally following Mendelian segregation.
Databáze: OpenAIRE
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