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CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of Coffea canephora

Abstract

Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps.

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Acknowledgements

The authors thank Prof. Kunling Chen (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) for providing the pJIT163 vector bearing the Cas9 sequence. This work was supported by Agropolis Fondation (Project ID 1403-026, Labex Agro: ANR-10-LABX-0001-01), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)/Cofecub (Comité Français d’Évaluation de la Coopération Universitaire et Scientifique avec le Brésil) (Project No. 407-2012 between Lavras Federal University-Brazil, Montpellier SupAgro-France, IRD and CIRAD in Montpellier).

Funding

Agropolis Fondation, Project ID 1403-026, Labex Agro: ANR-10-LABX-0001-01.

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Contributions

J-CB and RG carried out the bioinformatics analysis in collaboration with ACOBIOM (Montpellier, France). J-CB and LG made the constructs. J-CB, ED, LG and HE carried out the genetic transformation of C. canephora, and the plantlet phenotyping. J-CB and ED extracted DNA samples used by J-CB and CC to identify mutation. J-CB, HE, CC and PM designed the study, drew up the experimental design and implemented it. J-CB, RG, PM and HE wrote the article, which was approved by all the authors.

Corresponding author

Correspondence to Jean-Christophe Breitler.

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The authors declare that they have no conflict of interest.

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Communicated by Sergio J. Ochatt.

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Breitler, JC., Dechamp, E., Campa, C. et al. CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of Coffea canephora. Plant Cell Tiss Organ Cult 134, 383–394 (2018). https://doi.org/10.1007/s11240-018-1429-2

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Keywords

  • CRISPR/Cas9
  • Coffea canephora
  • Phytoene desaturase
  • Somatic embryogenesis
  • Targeted mutagenesis