SBIR Phase II: Generating HLB and canker resistant citrus trees using CRISPR/Cas9

该奖项的全部或部分资金来自《2021年美国救援计划法案》(公法117-2)。这个小企业创新研究(SBIR)第二阶段项目的更广泛影响是帮助美国柑橘行业减轻两种疾病的破坏性影响：黄龙病和柑橘溃烂病，这两种疾病都已导致佛罗里达州数百万棵柑橘树死亡，州政府损失数十亿美元的收入，并导致数千人失业。HLB还蔓延到其他州，包括加利福尼亚州、德克萨斯州、亚利桑那州和佐治亚州。这个第二阶段的项目将使用集群规则间隔短回文重复(CRISPR)精确育种技术生产非转基因、抗病的柑橘树。这些新品种将提振美国的柑橘产业，特别是在佛罗里达州，HLB和溃烂病已导致产量下降79%。拟议的项目旨在将效应器-互动组和CRISPR结合起来，为种植者创造一个具有商业价值的柑橘新品种。该项目利用了一项研究，该研究确定了与导致疾病发展的细菌效应蛋白相互作用的柑橘基因。该项目探索了多重CRISPR系统对病原体-柑橘相互作用的破坏，以及具有商业价值的柑橘品种的相关抗病潜力。该项目将产生编辑有希望的目标基因的柑橘植株，并在温室和田间试验中测试这些新品种的抗性和其他农艺性状。其方法和途径是构建多重CRISPR质粒，并通过原生质体转化和再生获得非转基因抗性柑橘品种。该测试协议要求嫁接将HLB接种到新品种上，以确认大规模实验和田间试验的抗性和无性系微繁殖。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2). The broader impact of this Small Business Innovation Research (SBIR) Phase II project is to help the US citrus industry mitigate the devastating effects of two diseases: Huanglongbing (HLB) and citrus canker, both of which have killed millions of citrus trees in Florida, cost the state billions in lost revenue, and caused the loss of thousands of jobs. HLB has also spread to other states including California, Texas, Arizona, and Georgia. This Phase II project will produce non-GMO, disease-resistant citrus trees using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) precision breeding technology. These new varieties will boost the US citrus industry, especially in Florida where HLB and canker have led to a 79% production decline. The proposed project aims to combine effector-interactome and CRISPR to create a commercially valuable new citrus variety for growers. This project leverages research that identified citrus genes that interact with bacterial effector proteins causing disease development. The project explores disruption of the pathogen-citrus interactions by a multiplex CRISPR system and the associated potential for disease resistance in commercially valuable citrus varieties. This project will generate citrus plants with edits to promising target genes and test those new varieties for resistance and other agronomic traits in greenhouse and field trials. The methods and approaches are multiplex CRISPR plasmid constructions, and citrus protoplast transformation and regeneration to create non-transgenic resistant citrus varieties. The testing protocol calls for grafting to inoculate the new varieties with HLB to confirm resistance and clonal micropropagation for large-scale experiments and field trials.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.