SBIR-STTR Award

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.

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.