title: The design and in vivo evaluation of engineered I-OnuI-based enzymes for HEG gene drive.
abstract: The homing endonuclease gene (HEG) drive system, a promising genetic approach for controlling arthropod populations, utilises engineered nucleases to spread deleterious mutations that inactivate individual genes throughout a target population. Previous work with a naturally occurring LAGLIDADG homing endonuclease (I-SceI) demonstrated its feasibility in both Drosophila and Anopheles. Here we report on the next stage of this strategy: the redesign of HEGs with customized specificity in order to drive HEG-induced 'homing' in vivo via break-induced homologous recombination. Variants targeting a sequence within the Anopheles AGAP004734 gene were created from the recently characterized I-OnuI endonuclease, and tested for cleavage activity and frequency of homing using a model Drosophila HEG drive system. We observed cleavage and homing at an integrated reporter for all endonuclease variants tested, demonstrating for the first time that engineered HEGs can cleave their target site in insect germline cells, promoting targeted mutagenesis and homing. However, in comparison to our previously reported work with I-SceI, the engineered I-OnuI variants mediated homing with a reduced frequency, suggesting that site-specific cleavage activity is insufficient by itself to ensure efficient homing. Taken together, our experiments take a further step towards the development of a viable HEG-based population control strategy for insects.
authors.affiliation: Department of Genetics, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Yuk-Sang
authors.initials: YS
authors.lastname: Chan
authors.forename: Ryo
authors.initials: R
authors.lastname: Takeuchi
authors.forename: Jordan
authors.initials: J
authors.lastname: Jarjour
authors.forename: David S
authors.initials: DS
authors.lastname: Huen
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
authors.forename: Steven
authors.initials: S
authors.lastname: Russell
mesh_terms: D000818:Animals; D000852:Anopheles / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005818:Genetic Engineering; D018095:Germ-Line Mutation; D059765:Homologous Recombination; D019476:Insect Proteins / Q000235:genetics*; D010572:Pest Control, Biological / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D019476:Insect Proteins; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; C066166:AI4 protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific
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