title: Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.
abstract: Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.
authors.affiliation: Institute for Insect Biotechnology, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany.
authors.forename: Irina
authors.initials: I
authors.lastname: Häcker
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, Maryland 20850, USA.|Insect Transformation Facility, University of Maryland, College Park, Rockville, Maryland 20850, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Harrell Ii
authors.affiliation: Mathematical Institute, Justus-Liebig-University Giessen, Arndtstrasse 2, 35392 Giessen, Germany.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Eichner
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, Maryland 20850, USA.
authors.forename: Kristina L
authors.initials: KL
authors.lastname: Pilitt
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Rockville, Maryland 20850, USA.|Department of Entomology, University of Maryland, College Park, Rockville, Maryland 20850, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: O'Brochta
authors.affiliation: USDA/ARS, Center for Medical, Agricultural and Veterinary Entomology, 1700 SW 23rd Drive, Gainesville, FL 32608, USA.
authors.forename: Alfred M
authors.initials: AM
authors.lastname: Handler
authors.affiliation: Institute for Insect Biotechnology, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany.
authors.forename: Marc F
authors.initials: MF
authors.lastname: Schetelig
mesh_terms: D000330:Aedes / Q000235:genetics* / Q000502:physiology / Q000821:virology; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites / Q000235:genetics; D005260:Female; D005298:Fertility / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D049750:Genome, Insect / Q000235:genetics*; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D008136:Longevity / Q000235:genetics; D008297:Male; D000072138:Mosquito Vectors / Q000235:genetics* / Q000502:physiology / Q000821:virology; D011995:Recombination, Genetic
chemical_list: C045073:Cre recombinase; D019426:Integrases