![]() ![]() The sequencing of the apple ( Velasco et al., 2010 Daccord et al., 2017) and pear ( Wu et al., 2013 Chagné et al., 2014) genomes has opened the way to the development of many genomic resources, which also increases the need for accurate tools of gene function analysis in these species. In this context, genetic engineering appears as a powerful tool to accelerate the improvement of existing apple and pear elite cultivars. In addition, most fruit trees are produced by clonal propagation, traditional cultivars are still dominant and the speed of introduction of new hybrid varieties on the market is slow. Conventional breeding of both species is limited by their long reproductive cycle and their high degree of heterozygosity. The world pear production in 2016 reached 27 millions tons, including both European pears ( Pyrus communis L.) and Asian pears (P. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines.Īpple ( Malus x domestica Bork.) is one of the major fruit crops produced in the world with a production over 89 million tons in 2016. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. ![]() These gRNAs were placed under the control of the U3 and U6 apple promoters. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. As a proof of concept, we chose to knock-out the Phytoene Desaturase ( PDS) and Terminal Flower 1 ( TFL1) genes. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. IRHS, INRA, Agrocampus-Ouest, Université d’Angers, SFR 4207 QuaSaV, Beaucouzé, France.Aurélie Charrier †, Emilie Vergne †, Nicolas Dousset, Andréa Richer, Aurélien Petiteau and Elisabeth Chevreau *
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