Single and multiple gene knockouts by CRISPR–Cas9 in maize

Doll, Nicolas M.; Gilles, Laurine M.; Gérentes, Marie-France; Richard, Christelle; Just, Jeremy; Fierlej, Yannick; Borrelli, Virginia M. G.; Gendrot, Ghislaine; Ingram, Gwyneth C.; Rogowsky, Peter M.; Widiez, Thomas

doi:10.1007/s00299-019-02378-1

Original Article
Published: 25 January 2019

Single and multiple gene knockouts by CRISPR–Cas9 in maize

Plant Cell Reports volume 38, pages 487–501 (2019)Cite this article

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Abstract

Key message

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.

Abstract

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.

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Data Availability

The two plasmids generated during the current study are available at GeneBank under the accession number MH662439 for L1609 and MH662440 for L1611.

Abbreviations

bp:: Base pairs
Cas9:: CRISPR-associated protein 9
CRISPR:: Clustered regularly interspaced short palindromic repeats
DSB:: Double-strand break
EMS:: Ethyl methanesulfonate
ESR:: Embryo surrounding region
HR:: Homologous recombination
MMEJ:: Microhomology-mediated end joining
NHEJ:: Non-homologous end joining
PAM:: Protospacer adjacent motif
SDN1:: Site-directed nuclease 1
sgRNA:: Single guide RNA
shRNA:: Short hairpin RNA, also referring to scaffold RNA
T-DNA:: Transfer DNA

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Acknowledgements

We thank Bing Yang (Iowa State University) for sharing the Iowa vectors prior to publication, Jean-Philippe Pichon (Biogemma SA) for providing unpublished genomic sequences of genotype A188, Justin Berger, Patrice Bolland and Alexis Lacroix for maize culture, Isabelle Desbouchages and Hervé Leyral for buffers and media preparation, as well as Sandrine Chaignon, Jérôme Laplaige and Edwige Delahaye for technical assistance. We acknowledge support by the Investissement d’Avenir program of the French National Agency of Research for the project GENIUS (ANR-11-BTBR-0001_GENIUS) to PMR and by the INRA Plant Science and Breeding Division for the project SeedCom to TW. NMD is funded by a PhD fellowship from the Ministère de l’Enseignement Supérieur et de la Recherche. LMG and YF are supported by CIFRE fellowships of the ANRT (Grants 2015/0777 and 2018/0480). VMGB is supported by the Doctoral School on the Agro-Food System (Agrisystem) of Università Cattolica del Sacro Cuore (Italy).

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Authors and Affiliations

Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, 69342, Lyon, France
Nicolas M. Doll, Laurine M. Gilles, Marie-France Gérentes, Christelle Richard, Jeremy Just, Yannick Fierlej, Ghislaine Gendrot, Gwyneth C. Ingram, Peter M. Rogowsky & Thomas Widiez
Limagrain Europe SAS, Research Centre, 63720, Chappes, France
Laurine M. Gilles
MAS Seeds, Route de Saint-Sever, 40280, Haut-Mauco, France
Yannick Fierlej
Department of Sustainable Crop Production, Università Cattolica del Sacro Cuore, Piacenza, Italy
Virginia M. G. Borrelli

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Nicolas M. Doll
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Laurine M. Gilles
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Marie-France Gérentes
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Christelle Richard
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Jeremy Just
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Yannick Fierlej
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Virginia M. G. Borrelli
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Ghislaine Gendrot
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Gwyneth C. Ingram
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Peter M. Rogowsky
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Thomas Widiez
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Correspondence to Thomas Widiez.

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Conflict of interest

LMG is employed by Limagrain Europe. YF is employed by MAS seed. PMR is part of the GIS-BV (“Groupement d’Intérêt Scientifique Biotechnologies Vertes”).

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Doll, N.M., Gilles, L.M., Gérentes, MF. et al. Single and multiple gene knockouts by CRISPR–Cas9 in maize. Plant Cell Rep 38, 487–501 (2019). https://doi.org/10.1007/s00299-019-02378-1

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Received: 31 August 2018
Accepted: 07 January 2019
Published: 25 January 2019
Issue Date: 01 April 2019
DOI: https://doi.org/10.1007/s00299-019-02378-1

Keywords

CRISPR
Gene editing
Maize
SDN1
Mutagenesis
Zea mays

Single and multiple gene knockouts by CRISPR–Cas9 in maize