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

• Published in: Plant cell, tissue and organ culture Vol. 134; no. 3; pp. 383 - 394
• Main Authors: Breitler, Jean-Christophe, Dechamp, Eveline, Campa, Claudine, Zebral Rodrigues, Leonardo Augusto, Guyot, Romain, Marraccini, Pierre, Etienne, Hervé
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.09.2018; Springer Nature B.V; Springer Verlag
• Subjects: Agronomy; Biomedical and Life Sciences; Coffea canephora; Coffee; CRISPR; Desaturase; Domestication; Editing; Embryonic growth stage; Embryos; Exons; Gene sequencing; Genes; Genetic transformation; Genome editing; Genomes; Germination; gRNA; Life Sciences; Mutation; Original Article; Plant breeding; Plant Genetics and Genomics; Plant Pathology; Plant Physiology; Plant Sciences; Regeneration; Ribonucleic acid; RNA; RNA editing; Site-directed mutagenesis; Somatic embryogenesis; Target recognition; Vegetal Biology; Targeted mutagenesis; CRISPR/Cas9; Phytoene desaturase; Somatic embryogenesis; Coffea canephora; CRISPR/CA9; Phytoene Desaturase
• ISSN: 0167-6857; 1573-5044
• DOI: 10.1007/s11240-018-1429-2

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.