@article{han_klobasa_oliveira_rotenberg_whitfield_lorenzen_2024, title={CRISPR/Cas9-mediated genome editing of <i>Frankliniella occidentalis</i>, the western flower thrips, via embryonic microinjection}, volume={4}, ISSN={["1365-2583"]}, DOI={10.1111/imb.12913}, abstractNote={Abstract The western flower thrips, Frankliniella occidentalis , poses a significant challenge in global agriculture as a notorious pest and a vector of economically significant orthotospoviruses. However, the limited availability of genetic tools for F. occidentalis hampers the advancement of functional genomics and the development of innovative pest control strategies. In this study, we present a robust methodology for generating heritable mutations in F. occidentalis using the CRISPR/Cas9 genome editing system. Two eye‐colour genes, white ( Fo‐w ) and cinnabar ( Fo‐cn ), frequently used to assess Cas9 function in insects were identified in the F. occidentalis genome and targeted for knockout through embryonic microinjection of Cas9 complexed with Fo‐w or Fo‐cn specific guide RNAs. Homozygous Fo‐w and Fo‐cn knockout lines were established by crossing mutant females and males. The Fo‐w knockout line revealed an age‐dependent modification of eye‐colour phenotype. Specifically, while young larvae exhibit orange‐coloured eyes, the colour transitions to bright red as they age. Unexpectedly, loss of Fo‐w function also altered body colour, with Fo‐w mutants having a lighter coloured body than wild type, suggesting a dual role for Fo‐w in thrips. In contrast, individuals from the Fo‐cn knockout line consistently displayed bright red eyes throughout all life stages. Molecular analyses validated precise editing of both target genes. This study offers a powerful tool to investigate thrips gene function and paves the way for the development of genetic technologies for population suppression and/or population replacement as a means of mitigating virus transmission by this vector.}, journal={INSECT MOLECULAR BIOLOGY}, author={Han, Jinlong and Klobasa, William and Oliveira, Lucas and Rotenberg, Dorith and Whitfield, Anna E. and Lorenzen, Marce D.}, year={2024}, month={Apr} }
 @article{wang_rivera_klobasa_lorenzen_2024, title={Evaluation of <i>Peregrinus maidis transformer-2</i> as a target for CRISPR-based control}, volume={19}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0295335}, abstractNote={The corn planthopper, Peregrinus maidis, is an economically important pest of corn and sorghum. Here we report the initial steps towards developing a CRISPR-based control method, precision guided sterile insect technique (pgSIT), for this hemipteran pest. Specifically, we evaluated the potential of transformer-2 (tra-2) as a target for sterilizing insects. First, we identified tra-2 transcripts within our P. maidis transcriptome database and performed RNA interference (RNAi) to confirm functional conservation. RNAi-mediated knockdown of Pmtra-2 in nymphs transformed females into pseudomales with deformed ovipositors resembling male claspers. While males showed no overt difference in appearance, they were indeed sterile. Importantly, the results were similar to those observed in another planthopper, Nilaparvata lugens. We also used CRISPR/Cas9 genome editing to assess the impact of tra-2 knockout in injectees. CRISPR-mediated knockout of Pmtra-2 had lethal effects on embryos, and hence not many injectees reached adulthood. However, mosaic knockout of Pmtra-2 did impact female and male fertility, which supports the use of tra-2 as a target for pgSIT in this hemipteran species.}, number={4}, journal={PLOS ONE}, author={Wang, Yu-Hui and Rivera, Dina Espinoza and Klobasa, William and Lorenzen, Marce D.}, year={2024}, month={Apr} }
 @article{wang_klobasa_chu_huot_whitfield_lorenzen_2023, title={Structural and functional insights into the ATP-binding cassette transporter family in the corn planthopper, Peregrinus maidis}, volume={32}, ISSN={0962-1075 1365-2583}, url={http://dx.doi.org/10.1111/imb.12840}, DOI={10.1111/imb.12840}, abstractNote={AbstractThe corn planthopper, Peregrinus maidis, is an economically important pest of maize and sorghum. Its feeding behaviour and the viruses it transmits can significantly reduce crop yield. The control of P. maidis and its associated viruses relies heavily on insecticides. However, control has proven difficult due to limited direct exposure of P. maidis to insecticides and rapid development of resistance. As such, alternative control methods are needed. In the absence of a genome assembly for this species, we first developed transcriptomic resources. Then, with the goal of finding targets for RNAi‐based control, we identified members of the ATP‐binding cassette transporter family and targeted specific members via RNAi. PmABCB_160306_3, PmABCE_118332_5 and PmABCF_24241_1, whose orthologs in other insects have proven important in development, were selected for knockdown. We found that RNAi‐mediated silencing of PmABCB_160306_3 impeded ovary development; disruption of PmABCE_118332_5 resulted in localized melanization; and knockdown of PmABCE_118332_5 or PmABCF_24241_1 each led to high mortality within five days. Each phenotype is similar to that found when targeting the orthologous gene in other species and it demonstrates their potential for use in RNAi‐based P. maidis control. The transcriptomic data and RNAi results presented here will no doubt assist with the development of new control methods for this pest.}, number={4}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Wang, Yu‐Hui and Klobasa, William and Chu, Fu‐Chyun and Huot, Ordom and Whitfield, Anna E. and Lorenzen, Marcé}, year={2023}, month={Apr}, pages={412–423} }
 @article{klobasa_chu_huot_grubbs_rotenberg_whitfield_lorenzen_2021, title={Microinjection of Corn Planthopper, <em>Peregrinus maidis</em>, Embryos for CRISPR/Cas9 Genome Editing}, volume={3}, ISSN={1940-087X}, url={http://dx.doi.org/10.3791/62417}, DOI={10.3791/62417}, abstractNote={The corn planthopper, Peregrinus maidis, is a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.}, number={169}, journal={Journal of Visualized Experiments}, publisher={MyJove Corporation}, author={Klobasa, William and Chu, Fu-Chyun and Huot, Ordom and Grubbs, Nathaniel and Rotenberg, Dorith and Whitfield, Anna E. and Lorenzen, Marcé D.}, year={2021}, month={Mar} }
 @article{chu_klobasa_grubbs_lorenzen_2017, title={Development and use of a piggyBac
-based jumpstarter system in Drosophila suzukii}, volume={97}, ISSN={0739-4462}, url={http://dx.doi.org/10.1002/arch.21439}, DOI={10.1002/arch.21439}, abstractNote={AbstractSpotted wing drosophila, Drosophila suzukii, is an invasive pest that primarily attacks fresh, soft‐skinned fruit. Although others have reported successful integration of marked piggyBac elements into the D. suzukii genome, with a very respectable transgenesis rate of ∼16%, here we take this work a step further by creating D. suzukii jumpstarter strains. These were generated through integration of a fluorescent‐marked Minos element carrying a heat shock protein 70‐driven piggyBac transposase gene. We demonstrate that there is a dramatic increase in transformation rates when germline transformation is performed in a transposase‐expressing background. For example, we achieved transformation rates as high as 80% when microinjecting piggyBac‐based plasmids into embryos derived from one of these D. suzukii jumpstarter strains. We also investigate the effect of insert size on transformation efficiency by testing the ability of the most efficient jumpstarter strain to catalyze integration of differently‐sized piggyBac elements. Finally, we demonstrate the ability of a jumpstarter strain to remobilize an already‐integrated piggyBac element to a new location, demonstrating that our jumpstarter strains could be used in conjunction with a piggyBac‐based donor strain for genome‐wide mutagenesis of D. suzukii.}, number={3}, journal={Archives of Insect Biochemistry and Physiology}, publisher={Wiley}, author={Chu, Fu-Chyun and Klobasa, William and Grubbs, Nathaniel and Lorenzen, Marcé D.}, year={2017}, month={Dec}, pages={e21439} }
 @article{chu_klobasa_wu_pinzi_grubbs_gorski_cardoza_lorenzen_2017, title={Germline transformation of the western corn rootworm, Diabrotica virgifera virgifera}, volume={26}, ISSN={0962-1075}, url={http://dx.doi.org/10.1111/imb.12305}, DOI={10.1111/imb.12305}, abstractNote={AbstractThe western corn rootworm (WCR), a major pest of maize, is notorious for rapidly adapting biochemically, behaviourally and developmentally to a variety of control methods. Despite much effort, the genetic basis of WCR adaptation remains a mystery. Since transformation‐based applications such as transposon tagging and enhancer trapping have facilitated genetic dissection of model species such as Drosophila melanogaster, we developed a germline‐transformation system for WCR in an effort to gain a greater understanding of the basic biology of this economically important insect. Here we report the use of a fluorescent‐marked Minos element to create transgenic WCR. We demonstrate that the transgenic strains express both an eye‐specific fluorescent marker and piggyBac transposase. We identified insertion‐site junction sequences via inverse PCR and assessed insertion copy number using digital droplet PCR (ddPCR). Interestingly, most WCR identified as transgenic via visual screening for DsRed fluorescence proved to carry multiple Minos insertions when tested via ddPCR. A total of eight unique insertion strains were created by outcrossing the initial transgenic strains to nontransgenic WCR mates. Establishing transgenic technologies for this beetle is the first step towards bringing a wide range of transformation‐based tools to bear on understanding WCR biology.}, number={4}, journal={Insect Molecular Biology}, publisher={Wiley}, author={Chu, F. and Klobasa, W. and Wu, P. and Pinzi, S. and Grubbs, N. and Gorski, S. and Cardoza, Y. and Lorenzen, M. D.}, year={2017}, month={Apr}, pages={440–452} }