@article{chu_wu_pinzi_grubbs_cohen_lorenzen_2023, title={An Optimized Small-Scale Rearing System to Support Embryonic Microinjection Protocols for Western Corn Rootworm, Diabrotica virgifera virgifera}, volume={14}, ISSN={2075-4450}, url={http://dx.doi.org/10.3390/insects14080683}, DOI={10.3390/insects14080683}, abstractNote={Western corn rootworm (WCR), a major pest of corn, has been reared in laboratories since the 1960s. While established rearing methods are appropriate for maintaining WCR colonies, they are not optimal for performing germline transformation or CRISPR/Cas9-based genome editing. Here we report the development of an optimized rearing system for use in WCR functional genomics research, specifically the development of a system that facilitates the collection of preblastoderm embryos for microinjection as well as gathering large larvae and pupae for downstream phenotypic screening. Further, transgenic-based experiments require stable and well-defined survival rates and the ability to manipulate insects at every life stage. In our system, the WCR life cycle (egg to adult) takes approximately 42 days, with most individuals eclosing between 41 and 45 days post oviposition. Over the course of one year, our overall survival rate was 67%. We used this data to establish a quality control system for more accurately monitoring colony health. Herein, we also offer detailed descriptions for setting up single-pair crosses and conducting phenotypic screens to identify transgenic progeny. This study provides a model for the development of new rearing systems and the establishment of highly controlled processes for specialized purposes.}, number={8}, journal={Insects}, publisher={MDPI AG}, author={Chu, Fu-Chyun and Wu, Pei-Shan and Pinzi, Sofia and Grubbs, Nathaniel and Cohen, Allen Carson and Lorenzen, Marcé D.}, year={2023}, month={Aug}, pages={683} }
 @article{dossey_oppert_chu_lorenzen_scheffler_simpson_koren_johnston_kataoka_ide_2023, title={Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture}, volume={13}, ISSN={2218-273X}, url={http://dx.doi.org/10.3390/biom13040589}, DOI={10.3390/biom13040589}, abstractNote={Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.}, number={4}, journal={Biomolecules}, publisher={MDPI AG}, author={Dossey, Aaron T. and Oppert, Brenda and Chu, Fu-Chyun and Lorenzen, Marcé D. and Scheffler, Brian and Simpson, Sheron and Koren, Sergey and Johnston, J. Spencer and Kataoka, Kosuke and Ide, Keigo}, year={2023}, month={Mar}, pages={589} }
 @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{oppert_dossey_chu_satovic-vuksic_plohl_smith_koren_olmstead_leierer_ragan_et al._2023, title={The Genome of the Yellow Mealworm, <i>Tenebrio molitor</i>: It's Bigger Than You Think}, volume={14}, ISSN={["2073-4425"]}, DOI={10.3390/genes14122209}, abstractNote={Background: Insects are a sustainable source of protein for human food and animal feed. We present a genome assembly, CRISPR gene editing, and life stage-specific transcriptomes for the yellow mealworm, Tenebrio molitor, one of the most intensively farmed insects worldwide. Methods: Long and short reads and long-range data were obtained from a T. molitor male pupa. Sequencing transcripts from 12 T. molitor life stages resulted in 279 million reads for gene prediction and genetic engineering. A unique plasmid delivery system containing guide RNAs targeting the eye color gene vermilion flanking the muscle actin gene promoter and EGFP marker was used in CRISPR/Cas9 transformation. Results: The assembly is approximately 53% of the genome size of 756.8 ± 9.6 Mb, measured using flow cytometry. Assembly was complicated by a satellitome of at least 11 highly conserved satDNAs occupying 28% of the genome. The injection of the plasmid into embryos resulted in knock-out of Tm vermilion and knock-in of EGFP. Conclusions: The genome of T. molitor is longer than current assemblies (including ours) due to a substantial amount (26.5%) of only one highly abundant satellite DNA sequence. Genetic sequences and transformation tools for an insect important to the food and feed industries will promote the sustainable utilization of mealworms and other farmed insects.}, number={12}, journal={GENES}, author={Oppert, Brenda and Dossey, Aaron T. and Chu, Fu-Chyun and Satovic-Vuksic, Eva and Plohl, Miroslav and Smith, Timothy P. L. and Koren, Sergey and Olmstead, Morgan L. and Leierer, Dewey and Ragan, Gail and et al.}, year={2023}, month={Dec} }
 @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{oppert_chu_reyna_pinzi_adrianos_perkin_lorenzen_2019, title={Effects of targeting eye color in
            Tenebrio molitor
            through RNA interference of tryptophan 2,3‐dioxygenase (
            vermilion
            ): Implications for insect farming}, volume={101}, ISSN={0739-4462 1520-6327}, url={http://dx.doi.org/10.1002/arch.21546}, DOI={10.1002/arch.21546}, abstractNote={Abstract}, number={1}, journal={Archives of Insect Biochemistry and Physiology}, publisher={Wiley}, author={Oppert, Brenda and Chu, Fu‐Chyun and Reyna, Steven and Pinzi, Sofia and Adrianos, Sherry and Perkin, Lindsey and Lorenzen, Marcé}, year={2019}, month={Mar}, pages={e21546} }
 @article{chu_wu_pinzi_grubbs_lorenzen_2018, title={Microinjection of Western Corn Rootworm, <em>Diabrotica virgifera virgifera</em>, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing}, ISSN={1940-087X}, url={http://dx.doi.org/10.3791/57497}, DOI={10.3791/57497}, abstractNote={The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR's genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.}, number={134}, journal={Journal of Visualized Experiments}, publisher={MyJove Corporation}, author={Chu, Fu-Chyun and Wu, Pei-Shan and Pinzi, Sofia and Grubbs, Nathaniel and Lorenzen, Marcé D.}, year={2018}, month={Apr} }
 @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} }