{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T10:17:31Z","timestamp":1788171451061,"version":"build-2803163510"},"reference-count":40,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T00:00:00Z","timestamp":1788134400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Secretariat for Science, Innovation and Technology of Panama","award":["FID18-050"],"award-info":[{"award-number":["FID18-050"]}]},{"award":["FID18-050"],"award-info":[{"award-number":["FID18-050"]}],"id":[{"id":"https:\/\/ror.org\/03gat5t60","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR\/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of \u2018Valencia\u2019 and hybrid genotypes (CsH1\u2013CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L\u22121 GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 \u00b5M) significantly enhanced rooting, increasing efficiencies to 37.1% in \u2018Valencia\u2019 and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR\/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure.<\/jats:p>","DOI":"10.3390\/plants15172664","type":"journal-article","created":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T09:21:19Z","timestamp":1788168079000},"page":"2664","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Dual-gRNA CRISPR\/Cas9 Deletion of CsDMR6 in Sweet Orange Supported by Improved In Vitro Regeneration"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-7712-3369","authenticated-orcid":false,"given":"Sandra","family":"Sopalda","sequence":"first","affiliation":[{"name":"Master\u2019s Program in Agricultural Sciences, Faculty of Agricultural Sciences, University of Chile, Santiago 8820808, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4546-3247","authenticated-orcid":false,"given":"Ricardo","family":"Vergara","sequence":"additional","affiliation":[{"name":"Biotechnology Laboratory, Institute of Agricultural Research\u2013La Platina, Santiago 8831314, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marisol","family":"Mu\u00f1oz","sequence":"additional","affiliation":[{"name":"Biotechnology Laboratory, Institute of Agricultural Research\u2013La Platina, Santiago 8831314, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0055-4551","authenticated-orcid":false,"given":"Carlos","family":"Aguirre","sequence":"additional","affiliation":[{"name":"Biotechnology Laboratory, Institute of Agricultural Research\u2013La Platina, Santiago 8831314, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos","family":"Mu\u00f1oz","sequence":"additional","affiliation":[{"name":"Plant Breeding Laboratory, Faculty of Agricultural Sciences, University of Chile, Santiago 8820808, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9013-1906","authenticated-orcid":false,"given":"Humberto","family":"Prieto","sequence":"additional","affiliation":[{"name":"Biotechnology Laboratory, Institute of Agricultural Research\u2013La Platina, Santiago 8831314, Chile"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Ghosh, D., Kokane, S., Savita, B.K., Kumar, P., Sharma, A.K., Ozcan, A., Kokane, A., and Santra, S. 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