In a first study, we succeeded in creating targeted mutagenesis by CRISPR-Cas9 (Charrier et al, 2019). We optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS, albino phenotype) and Terminal Flower 1 (TFL1, early flowering phenotype) genes. To improve the edition efficiency, two different guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the MdU3 and MdU6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. 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. 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. In most cases, Cas9 nuclease cut the DNA within the twenty targeted base pairs near the PAM (protospacer adjacent motif) and insertions were more frequent than deletions or substitutions. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. 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.

In a second study, we succeeded in developing targeted base conversion by CRISPR-Cas9 base editing (Malabarba et al, 2020). We demonstrated this feasibility in apple and pear using two easily scorable genes: acetolactate synthase-ALS (conferring resistance to chlorsulfuron) and PDS (albino phenotype). The two guide RNAs were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9 (catalytically impaired, only one of the two strands is cleaved). Using this vector, we induced C-to-T DNA substitutions in the target genes, leading to discrete variations in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes, we successfully obtained chlorsulfuron resistant and albino lines in pear (figure on pear). Overall, our work indicates that base editing can be applied to create accurate genome edits in perennial plants.

In the next two years, the team intends to develop the latest tool to date derived from CRISPR technology: prime-editing, which makes it possible to replace a complete sequence of 7-21 bases instead of a single base of the DNA sequence. Prime editing directly adds the new desired sequence into the target site using a Cas9 nickase fused to an engineered reverse transcriptase. This fusion enzyme is driven by a special guide RNA (prime editing guide RNA) that both specifies the target site and encodes the desired 7-21 bases to add. Until now, no work is available with this method on perennial plants. 

Note: the genes targeted in these studies (herbicide resistance, albino phenotype, early flowering) are chosen for their easily identifiable phenotype in the laboratory. For the ResPom team, they do not represent agronomic development targets in the field for these species.

Associated publications:

Charrier A, Vergne E, Dousset N, Richer A, Petiteau A, Chevreau E. Efficient Targeted Mutagenesis in Apple and First Time Edition of Pear Using the CRISPR-Cas9 System. Front Plant Sci. 2019 Feb 6;10:40. doi: 10.3389/fpls.2019.00040. PMID: 30787936; PMCID: PMC6373458.

Malabarba J, Chevreau E, Dousset N, Veillet F, Moizan J, Vergne E. New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing. Int J Mol Sci. 2020 Dec 30;22(1):319. doi: 10.3390/ijms22010319. PMID: 33396822; PMCID: PMC7795782.