ENABLING EFFICIENT TRANSGENE-FREE PRECISE GENE EDITING IN CARROT

Objective 1: Develop efficient RNP delivery systems for targeted mutagenesis in carrotTo achieve efficient targeted mutagenesis in carrot, optimized Cas12a RNP delivery systems will first be established in carrot protoplasts. Three Cas12a of high editing performance will be used, including LbCas12a, LbCas12a-Ultra and AsCas12a-Ultra. Four genes will be chosen as the targets: 1) The phytoene desaturase gene (DcPDS) is a rate-limiting enzyme in carotenoid synthesis. Knockout of DcPDS will result in a photo-bleached phenotype that allows us to visualize targeted mutagenesis in carrot; 2) The lycopene epsilon-cyclase gene (DcLCYE) catalyzes the conversion from lycopene to α-carotene. Knockout of DcLCYE will shift lycopene metabolism toward the synthesis of β-carotene, provitamin A; 3) Dau c 1.01 and Dau c 1.02 are two genes encoding pathogenesis-related protein-10 (PR10) isoforms causing allergic reactions of carrots. Simultaneous knockout of these two genes can potentially create hypoallergenic carrots. Two crRNAs will be designed for each target gene using CRISPR-DT. The crRNAs will be preferred to locate near the 5'-terminus of coding sequences to have a higher chance to disrupt gene functions. The crRNAs with high predicted editing efficiencies and low potential off-targets will be used. All crRNAs will be synthesized as single-stranded RNAs with end protection and used for RNP delivery. Our first approach to obtain stably edited carrot plants is to regenerate from edited protoplasts. Our second approach to obtain stably edited carrot plants is to deliver Cas12a RNP into carrot calli. In both approaches, the crRNAs with the highest editing efficiencies for each target gene, along with the best performing Cas12a, will be used for RNP transformation. The Cas12a/crRNAs for Dau c 1.01 and Dau c 1.02 will be simultaneously delivered into protoplasts for a double knockout. For DcPDS or DcLCYE target gene, the best crRNA will be chosen for single target editing. The transformed protoplasts or calli will be maintained for plant regeneration, followed by genotyping of targeted mutants in the T0 generation.Objective 2: Develop an efficient HDR system with delivery of Cas12a/crRNA RNP and oligo donorsTo establish a transgene-free Cas12a-mediated HDR method in carrot, we will first conduct a series of experiments in carrot protoplasts to optimize the system. As a proof of concept, we will insert a small epitope tag, a 6XHis tag, to the C-terminus of DcPDS. Since PDS is a rate-limiting enzyme in carotenoid synthesis, a polyhistidine tag fusion is useful for studying PDS regulation. For example, it will allow us to quantify the PDS level at any circumstance, such as under biotic or abiotic stresses, or when other genes are up or down regulated, thus help us understand the regulatory network of carotenoid biosynthesis. The crRNA will be designed to locate at the C-terminus of the coding sequence. The optimized RNP delivery system obtained in the first objective will be used here. Donor sequence will harbor the inserted 6XHis tag sequence, as well as synonymous mutations to disrupt the crRNA targeting sequence or the PAM sequence, so that once the donor is incorporated into the genome, it will not be cut by Cas12a again. Multiple factors including dosage of oligo donors, donor strand, length of donors, and donor end protection and modification will be assessed to identify an optimized condition for highest HDR efficiency based on RNP delivery of Cas12a. The donor dosage and format that lead to the highest HDR efficiency will be applied to generate stably edited carrot plants, using the similar approaches described in the first objective. Since lower editing efficiency is expected for HDR-mediated precise genome editing, the transformation scale will be increased. Transformed protoplasts will be used to regenerate carrot plants, followed by genotyping of HDR events in the T0 generation.