Malus genome editing via CRISPR/CAS9 to develop sustainable and pest free apples

Most commercial apples are sensitive to pathogens causing re blight (Erwinia amylovora) and powdery mildew (Podosphaera leucotricha). The control of these pathogens requires pesticides and eradication with a negative impact on the environment and huge economic losses. Resistant varieties were obtained by classical breeding via the transfer of resistance genes (R-genes) from wild apples, but this approach is time-consuming, leads to low-quality plants, and the adjunct resistance is not durable. The suppression of susceptibility genes (S-genes) could be used to confer durable resistance to apples but past attempts to knock-down S-genes were mostly transgenic-based thus limiting the use of resulting plants. The aim of this project is to use CRISPR/Cas9 to develop pest-free apples by simultaneously mutating S-genes responsible for the susceptibility to E. amylovora and P. leucotricha. Gene mutation is obtained through the delivery of a vector containing the editing machinery combined with a heat-inducible recombination system to permit the elimination of vector DNA from the genome of plants after editing. Here we present the results obtained from the Agrobacterium-mediated delivery of this heat-excisable editing system. More than 50 Gala and Golden Delicious lines with no evident growth deciencies were regenerated and conrmed for T-DNA integration by PCR. Single-pass sequencing indicated that about 80 % of such lines were edited in at least one of the four target genes and 30 % had all the four genes edited. Preliminary Illumina sequencing data (19 lines) revealed that editing events were mostly constituted by small deletions (<6 bp). Plants edited for single and multiple genes are planned to be scored for re blight and powdery mildew resistance and then to be submitted to heat-treatments for the excision of the editing cassette. A protoplast-to-plant regeneration procedure will also be developed during the project to allow DNA-free genome editing.