1. Please state project objectives and what work was done this quarter to address them: The purpose of this project is to generate non-transgenic HLB resistant Valencia and Hamlin sweet orange plants using CRISPR-Cas technology. Objective 1. Generate non-transgenic HLB resistant/tolerant Valencia and Hamlin sweet orange plants by mutation of HLB susceptibility genes. In total, six putative S genes will be edited. Constructs needed for CRISPR genome editing are being made. Multiple edited lines were generated for ACD2 gene. However, further confirmation demonstrated none of them was biallelic/homozygous mutant. We are genenerating more lines for ACD2 and other target genes. To speed up the process, we have developed an efficient co-editing strategy for generating transgene-free, gene-edited plants via Agrobacterium-mediated transient expression of cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP in planta. Specifically, CBE/gRNA was used to base edit the ALS gene to confer resistance to herbicide chlorsulfuron as a selection marker, which has no negative effects on plant phenotypes; Cas12a/crRNA was used for editing genes(s) of interest; GFP was used for selecting transgene-free transformants. Using this approach, transgene-free genome-edited plants can be relatively easily generated for citrus in the T0 generation. Whole genome sequencing further confirmed transgene-free and absence of off-target mutations in the edited plants. We are also using this strategy for genome editing of our target genes. Among the 8 target genes, we have done transformation again on 5/18, 5/25, or 6/25. The transformants are under regeneration. Objective 2. Generate cisgenic genome modified Valencia and Hamlin sweet orange plants by knock-in the gene encoding MaSAMP from Microcitrus. We are optimizing the knock-in method using the CRISPR technology. We have made some progress in knock-in methodology using non-transgenic approach. We have conducted multiple transformations useing the developed method for knockin with MaSAMP. The protoplasts were being regenerated.We have demonstrated the knock-in works using GFP as an insert. We have conducted knock-in for MaSAMP on 5/18, which is under regeneration. 2. Please state what work is anticipated for next quarter:To generate more genome edited lines for ACD2 and other target genes using both RNP method and the co-editing method. Conductu regeneration for all the transformants. 3. Please state budget status (underspend or overspend, and why):On schedule.