ets for CRISPR/Cas9-mediated gene drive in Bemisia tabaci

The goal of our BARD proposal was to build both the necessary infrastructure and knowledge for using the CRISPR/Cas9-based gene drive system to control the whitefly Bemisia tabaci. Our research focused on achieving three main goals: (1) establishing a CRISPR/Cas9 gene-editing system for producing genetically-edited B. tabaci; (2) generating and testing CRISPR/Cas9-mediated mutations targeting genes that represent two gene drive strategies: population replacement and population suppression; (3) using computer modeling to optimize strategies for applying CRISPR/Cas9 to control B. tabaci populations in the field. CRISPR gene drive is one of the most promising strategies for diminishing the negative impacts of harmful insects. This technique can introduce mutations into wild populations of pests that reduce their ability to cause damage, reduce their population size, or both. In principle, this can be selfsustaining because mutations carried by relatively few insects can increase in frequency and spread quickly throughout wild populations. Because of this sustainability and the potential benefits to society, agricultural gene-drive systems are most likely to be funded by government agencies, foundations, and grower associations; as with sterile insect releases and most biocontrol programs. Although gene drives have received intensive study in Drosophila and mosquito vectors of human disease, we were one of the first teams pursuing this approach for crop pests. Our project was also one of the first to address CRISPR gene drive in the Hemiptera, an insect order that includes hundreds of pest species. We focused on developing and implementing CRISPR gene drive to reduce the massive damage caused by B. tabaci. This haplodiploid insect is one of the world's most devastating crop pests. Whereas extensive work by others explored CRISPR in diploid species, our project pioneered application of this revolutionary technology to haplodiploids, which have a distinct system of inheritance that presents special challenges and opportunities. Our project has achieved several breakthroughs, including publication of the first paper analyzing CRISPR gene drive in haplodiploids (Li et al. 2020, see next section). Our modeling results from this landmark study demonstrate that CRISPR gene drive can work in haplodiploids, especially if fitness costs associated with the driver allele are low or nil. Our paper was the first to provide a conceptual framework for evaluating and optimizing CRISPR gene drive strategies for managing B. tabaci and other haplodiploid pests. Our breakthroughs in the laboratory have created the infrastructure needed to develop CRISPR for controlling B. tabaci. We established a microinjection system enabling us to introduce CRISPR-derived mutations into B. tabaci embryos. We have used this system to generate and track inherited eye-color mutants of B. tabaci. We have identified and cloned germline promoters, and demonstrated their function in transgenic B. tabaci embryos and other hemipteran insects. We have also developed a tool to easily identify B. tabaci harboring CRISPR-mediated mutations by tagging target genes using a transgenic fluorescent marker. The successful completion of our project provides all the knowledge and infrastructure essential for developing a novel genetic approach for B. tabaci control, which can serve as a non-chemical "green" alternative for managing this global pest. We predict that our discoveries will accelerate the development of the CRISPR gene drive technique for reducing the numbers of this pest and the damage it causes. Still, realization of the benefits of gene-drive technology for pest control will require sustained attention to potential environmental and societal impacts, as well as regulatory and implementation challenges. Given the great promise of this technology and the urgent need for better control methods, we expect that guidance documents and regulations will be in place to allow the scientific community to safely move gene drives for pest control from the laboratory to field trials.