@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={Abstract}, 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={Abstract}, 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={Abstract}, 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} }