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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2024)

The management of crop pests and invasive species presents a significant global challenge in food production, exacerbated by climate change and increased species mobility. Traditional non-specific pesticides, utilized for centuries, now pose environmental risks and encounter diminishing efficacy against evolving insect resistance. Consequently, there is a pressing need for safer, species-specific alternatives. In addressing this need, the first aim of my thesis was to implement the precision-guided sterile insect technique (pgSIT) for combatting the prevalent crop pest, Drosophila suzukii. With pgSIT, only sterile Drosophila suzukii males emerge from the population, which can be released into the wild to compete with the wild-type males for population suppression purposes. Our findings underscored the potential of using pgSIT as a safe, economically viable, and scalable approach to managing Drosophila suzukii populations. Additionally, to enhance the efficiency of existing mass-rearing strategies, my secondary aim was to develop a precise, rapid sex-sorting method using genetic techniques that can be used in a wide range of insect species. Leveraging fluorescence, I developed a sex-sorting technique, SEPARATOR, based on the transformer (tra) intron. This technique demonstrated efficacy across two insect species: Drosophila melanogaster, and Drosophila suzukii. The SEPARATOR allows female- or male-specific selection at different developmental stage using fluorescence markers, which opens up the sexing window and enable automatic sex-sorting, thereby allowing mass rearing of the genetically engineered insects. Finally, the last aim of my thesis was to develop an easily transferable and resistant-proof female-killing biocontrol technology. With this aim, we developed MitoFail (Mitochondrial female-associated lethality). This technique leverages the universally active restriction endonucleases shredding mitochondrial DNA molecules (mtDNA) only in females. By combining this with an inducible system, we were able to control female lethality with either temperature or doxycycline, providing a robust an adaptable biocontrol strategy.

    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2021)
    Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for mass release. While effective at generating F1 sterile males, pgSIT requires a genetic cross between the two parental strains, which requires maintenance and sexing of two strains in a factory. Therefore, to advance pgSIT further by removing this crossing step, here we describe a next-generation temperature-inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true breeding strain for TI-pgSIT that eliminates the requirement for sex sorting-a feature that may help further automate production at scale.
      Cover page: Temperature-Inducible Precision-Guided Sterile Insect Technique
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2020)
      Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance.
        Cover page: A drug-inducible sex-separation technique for insectsCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2020)
        Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.
          Cover page: Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple GenesCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2021)
          Homing-based gene drives, engineered using CRISPR/Cas9, have been proposed to spread desirable genes throughout populations. However, invasion of such drives can be hindered by the accumulation of resistant alleles. To limit this obstacle, we engineer a confinable population modification home-and-rescue (HomeR) drive in Drosophila targeting an essential gene. In our experiments, resistant alleles that disrupt the target gene function were recessive lethal and therefore disadvantaged. We demonstrate that HomeR can achieve an increase in frequency in population cage experiments, but that fitness costs due to the Cas9 insertion limit drive efficacy. Finally, we conduct mathematical modeling comparing HomeR to contemporary gene drive architectures for population modification over wide ranges of fitness costs, transmission rates, and release regimens. HomeR could potentially be adapted to other species, as a means for safe, confinable, modification of wild populations.
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2023)
            Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique.
              Cover page: Next-generation genetic sexing strain establishment in the agricultural pest Ceratitis capitata.Creative Commons 'BY' version 4.0 license
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2021)
              Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2019)
                  • Article
                  • Peer Reviewed
                  UC Berkeley Previously Published Works (2019)
                  The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed precision guided SIT (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. Here, to demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated sterile males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to potentially transform our ability to control insect agricultural pests and disease vectors.
                    • Article
                    • Peer Reviewed
                    UC San Diego Previously Published Works (2021)
                    The development of an extensive toolkit for potential point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect SARS-CoV-2. Herein, we outline the development of an alternative CRISPR nucleic acid diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens XPD3002 (CasRx) to detect SARS-CoV-2, an approach we term SENSR (sensitive enzymatic nucleic acid sequence reporter) that can detect attomolar concentrations of SARS-CoV-2. We demonstrate 100% sensitivity in patient-derived samples by lateral flow and fluorescence readout with a detection limit of 45 copy/μL. This technology expands the available nucleic acid diagnostic toolkit, which can be adapted to combat future pandemics.
                      Cover page: Development of a Rapid and Sensitive CasRx-Based Diagnostic Assay for SARS-CoV‑2
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