Improvement of crispr/CASg knock-out and base editing techniques in Apple and Pear

International audienceCRISPR/Cas9 has become the golden technique for gene knock-out in plants. In addition to gene knock-out through the generation of INDELsmutations, base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Our previous results with CRISPR/Cas9 in apple indicated the frequent production of phenotypic and edition chimeras, after edition of theeasily scorable gene phytoene desaturase (PDS). Therefore our rst goal was to determine if adding an adventitious regeneration step from leaves of the T0 plants, would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89 % werehomogeneous albino. Furthermore, the target zone sequences of twelve of these regenerated lines (T1) were studied and compared to the T0 sequences. The results showed that 99% of the T1 alleles were predicted to producing a truncated target protein and that 67% of T1 plants had less heterogeneous editing proles than the T0. This indicates that a regeneration step can efficiently reduce the initial chimerism. The second objective was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: PDS (conferring albino and dwarfphenotype by impaired chlorophyll and gibberellin synthesis) and acetolactate synthase (ALS) (conferring resistance to chlorsulfuron). The two guideRNAs (PDS+ALS) under MdU3 and MdU6 promoters, respectively, were coupled in the pDenCas9_PmCDA1_UGI vector which has a cytidine deaminase fused to a nickase Cas9. Using this cytidine-base editor, we precisely induced DNA substitutions in the target genes, leading to discrete variation in theamino-acid sequence and generating a loss-of-function allele. The successful application of base editing in the apple and pear creates new possibilities for genome engineering to explore desirable agronomic traits in these species