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 were proposed to be edited and we have lately added more targets. 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 further optimized the transgene-free CRISPR genome editing using Cas12a/crRNA ribonucleoprotein. 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. This has been published by Nat. Plants (9, 15911597, 2023. https://doi.org/10.1038/s41477-023-01520-y). Among the 8 target genes, we have done transformation again on 5/18, 5/25, 6/25, 7/27, and 8/17. 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, and 8/17 which are 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.