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

这个小企业创新研究（SBIR）第一阶段项目的更广泛的影响/商业潜力是设计对柑橘树中两种细菌性疾病的抗性，黄龙病（HLB）和柑橘溃疡病。 HLB和溃疡病使种植者损失了数十亿美元的树木被毁，并正在摧毁佛罗里达的柑橘产业。在佛罗里达，柑橘年产量已从2003-2004年生长季的2.42亿箱降至2017-2018年的4500万箱。 此外，在佛罗里达98%的柑橘生产区中可以发现柑橘溃疡病。与佛罗里达大学合作，该项目的目标是利用CRISPR/Cas9基因编辑来推进自然选择，使柑橘树同时抵抗HLB和柑橘溃疡病。如果成功，这种方法可以用来保护全球9300万吨柑橘产量。该SBIR项目的智力价值是通过使用CRISPR/Cas9的基因编辑来开发HLB和抗溃疡的柑橘品种。佛罗里达的柑橘种植者需要一个解决这些疾病的办法，而生产一种具有遗传抗性的柑橘品种是一个有希望的策略。这种方法是修改植物基因，这些基因是致病细菌分泌的蛋白质的目标。第一个目标是在商业上可行的柑橘品种中进行基因编辑。这将通过在柑橘原生质体中转化非整合的质粒或纯化的Cas9蛋白和体外转录的sgRNA来非转基因地完成。为了鉴定基因编辑，原生质体将生长到愈伤组织中，其将通过PCR和DNA异源双链消化以及桑格和/或高通量测序进行筛选。第二个目标将是采取具有确认的编辑的植物，克隆繁殖它们，并进行HLB或溃疡病抗性的初步测试。将用致病细菌接种植物，然后通过观察物理特性和通过PCR测量细菌滴度来监测疾病进展。在SBIR第二阶段，第一阶段中表现最好的突变将被组合并用于更广泛的温室和田间测试。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is to engineer resistance to two bacterial diseases in citrus trees, Huanglongbing (HLB) and citrus canker. HLB and canker have cost growers billions of dollars in destroyed trees and are decimating the Florida citrus industry. In Florida, annual citrus production has fallen from 242 million boxes in the 2003-2004 growing season to 45 million boxes in 2017-2018. Further, citrus canker may be found in 98% of the citrus production regions in Florida. Working with the University of Florida, the goal of this project is to use CRISPR/Cas9 gene editing to advance natural selection to make citrus trees resistant to both HLB and citrus canker. If successful, this approach could be used to protect the 93 million tons of citrus production worldwide. The intellectual merit of this SBIR project is to develop HLB and canker-resistant citrus varieties by using gene editing with CRISPR/Cas9. Florida citrus growers need a solution to these diseases, and producing a genetically-resistant citrus variety is a promising strategy. The approach is to modify plant genes that are the targets of proteins secreted by the disease-causing bacteria. The first objective is to conduct the gene editing in commercially viable citrus varieties. This will be accomplished non-transgenically by transforming a non-integrated plasmid or purified Cas9 protein and in vitro transcribed sgRNA in citrus protoplasts. To identify gene edits, protoplasts will be grown into callus, which will be screened by PCR and DNA heteroduplex digestion as well as Sanger and/or high-throughput sequencing. The second objective will be to take plants with confirmed edits, clonally propagate them, and conduct preliminary testing for HLB or canker resistance. Plants will be inoculated with the disease-causing bacteria and then monitored for disease progression by observation of physical characteristics and measurement of bacterial titers by PCR. The best performing mutations from Phase I will be combined and used for more extensive greenhouse and field testing during SBIR Phase II.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.