Publications by authors named "Xuechun Feng"

8 Publications

  • Page 1 of 1

Evaluation of Gene Knockouts by CRISPR as Potential Targets for the Genetic Engineering of the Mosquito .

CRISPR J 2021 08 19;4(4):595-608. Epub 2021 Jul 19.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, USA; University of Monash, Clayton, Australia.

mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people and, separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations. Yet, research into has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, in the sex-determination pathway and , a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily scored transgenesis marker.
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http://dx.doi.org/10.1089/crispr.2021.0028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392076PMC
August 2021

G-Protein Coupled Receptors (GPCRs): Signaling Pathways, Characterization, and Functions in Insect Physiology and Toxicology.

Int J Mol Sci 2021 May 17;22(10). Epub 2021 May 17.

Department of Biology Sciences, University of California, San Diego, CA 92093, USA.

G-protein-coupled receptors (GPCRs) are known to play central roles in the physiology of many organisms. Members of this seven α-helical transmembrane protein family transduce the extracellular signals and regulate intracellular second messengers through coupling to heterotrimeric G-proteins, adenylate cyclase, cAMPs, and protein kinases. As a result of the critical function of GPCRs in cell physiology and biochemistry, they not only play important roles in cell biology and the medicines used to treat a wide range of human diseases but also in insects' physiological functions. Recent studies have revealed the expression and function of GPCRs in insecticide resistance, improving our understanding of the molecular complexes governing the development of insecticide resistance. This article focuses on the review of G-protein coupled receptor (GPCR) signaling pathways in insect physiology, including insects' reproduction, growth and development, stress responses, feeding, behaviors, and other physiological processes. Hormones and polypeptides that are involved in insect GPCR regulatory pathways are reviewed. The review also gives a brief introduction of GPCR pathways in organisms in general. At the end of the review, it provides the recent studies on the function of GPCRs in the development of insecticide resistance, focusing in particular on our current knowledge of the expression and function of GPCRs and their downstream regulation pathways and their roles in insecticide resistance and the regulation of resistance P450 gene expression. The latest insights into the exciting technological advances and new techniques for gene expression and functional characterization of the GPCRs in insects are provided.
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http://dx.doi.org/10.3390/ijms22105260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156084PMC
May 2021

Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes.

Nat Commun 2021 05 20;12(1):2960. Epub 2021 May 20.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.

Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex quinquefasciatus and Drosophila melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.
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http://dx.doi.org/10.1038/s41467-021-23239-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137705PMC
May 2021

Multiple cytochrome P450 genes: conferring high levels of permethrin resistance in mosquitoes, Culex quinquefasciatus.

Sci Rep 2021 04 27;11(1):9041. Epub 2021 Apr 27.

Department of Entomology and Plant Pathology 301 Funchess Hall, Auburn University, Auburn, AL, 36849-5413, USA.

Insecticides, especially pyrethroids, are the most important in the insect pest control and preventing insect vector-borne human diseases. However, insect pests, including mosquitoes, have developed resistance in the insecticides that used against them. Cytochrome P450s are associated with insecticide resistance through overexpression and detoxification mechanisms in insect species. In this study, we utilized a powerful tool, the RNAi technique, to determine the roles of key P450 genes overexpressed in permethrin resistant mosquitoes that confer insecticide resistance to unravel the molecular basis of resistance mechanisms in the mosquito Culex quinquefasciatus. The results showed that knockdown of 8 key P450 genes using RNAi techniques significantly decreased resistance to permethrin in resistant mosquitoes. In silico modeling and docking analysis further revealed the potential metabolic function of overexpressed P450 genes in the development of insecticide resistance in mosquitoes. These findings not only highlighted the functional importance of these P450 genes in insecticide resistance, but also revealed that overexpression of multiple P450 genes was responsible for the high levels of insecticide resistance in a mosquito population of Culex quinquefasciatus.
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http://dx.doi.org/10.1038/s41598-021-88121-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079677PMC
April 2021

Functional Analyses of House Fly Carboxylesterases Involved in Insecticide Resistance.

Front Physiol 2020 16;11:595009. Epub 2020 Oct 16.

Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, United States.

Carboxylesterase-mediated metabolism is one of major mechanisms involved in insecticide resistance. Our previous study has identified multiple carboxylesterase genes with their expression levels were significantly upregulated in pyrethroid resistant house flies. To further explore their metabolic functions, we used insect (9) cells to express these carboxylesterases and measure their hydrolytic activities toward esterase substrates. Our results indicated that these carboxylesterases can efficiently hydrolyze α-naphthyl acetate rather than β- naphthyl acetate. A cell based MTT cytotoxicity assay indicated that carboxylesterase-expressing cells show enhanced tolerance to permethrin, suggesting important roles of these carboxylesterases in metabolizing permethrin and thereby protecting cells from permethrin treatments. The metabolic functions of carboxylesterases were further verified by conducting metabolism studies toward permethrin and its potential metabolites 3-phenoxybenzyl alcohol and 3-phenoxybenzaldehyde, which not only suggested the potential metabolic pathway of permethrin in insects, but also important roles of these candidate carboxylesterases in metabolizing permethrin and conferring resistance in house flies. Homology modeling and docking were finally conducted to reflect interactions between permethrin ligand and carboxylesterase proteins, visually confirming the metabolic functions of carboxylesterases to insecticides in house flies.
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http://dx.doi.org/10.3389/fphys.2020.595009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596742PMC
October 2020

A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment.

Nat Commun 2020 01 17;11(1):352. Epub 2020 Jan 17.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, 92093, USA.

CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.
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http://dx.doi.org/10.1038/s41467-019-13977-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969112PMC
January 2020

Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica.

J Vis Exp 2018 08 23(138). Epub 2018 Aug 23.

Department of Entomology and Plant Pathology, Auburn University;

Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of carboxylesterases. Three example experiments are presented: (1) expression and isolation of carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of carboxylesterases toward permethrin. The carboxylesterase gene MdαE7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (MdαE7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of MdαE7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of MdαE7 toward permethrin, directly indicating the involvement of carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.
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http://dx.doi.org/10.3791/58106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231707PMC
August 2018

Carboxylesterase genes in pyrethroid resistant house flies, Musca domestica.

Insect Biochem Mol Biol 2018 01 14;92:30-39. Epub 2017 Nov 14.

Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, USA. Electronic address:

Carboxylesterases are one of the major enzyme families involved in the detoxification of pyrethroids. Up-regulation of carboxylesterase genes is thought to be a major component of insecticide resistant mechanisms in insects. Based on the house fly transcriptome and genome database, a total of 39 carboxylesterase genes of different functional clades have been identified in house flies. In this study, eleven of these genes were found to be significantly overexpressed in the resistant ALHF house fly strain compared with susceptible aabys and wild-type CS strains. Eight up-regulated carboxylesterase genes with their expression levels were further induced to a higher level in response to permethrin treatments, indicating that constitutive and inductive overexpression of carboxylesterases are co-responsible for the enhanced detoxification of insecticides. Spatial expression studies revealed these up-regulated genes to be abundantly distributed in fat bodies and genetically mapped on autosome 2 or 3 of house flies, and their expression could be regulated by factors on autosome 1, 2 and 5. Taken together, these results demonstrate that multiple carboxylesterase genes are co-upregulated in resistant house flies, providing further evidence for their involvement in the detoxification of insecticides and development of insecticide resistance.
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http://dx.doi.org/10.1016/j.ibmb.2017.11.007DOI Listing
January 2018
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