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The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive

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 homin...

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Autores principales: Chan, Yuk-Sang, Takeuchi, Ryo, Jarjour, Jordan, Huen, David S., Stoddard, Barry L., Russell, Steven
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769382/
https://www.ncbi.nlm.nih.gov/pubmed/24040217
http://dx.doi.org/10.1371/journal.pone.0074254
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author Chan, Yuk-Sang
Takeuchi, Ryo
Jarjour, Jordan
Huen, David S.
Stoddard, Barry L.
Russell, Steven
author_facet Chan, Yuk-Sang
Takeuchi, Ryo
Jarjour, Jordan
Huen, David S.
Stoddard, Barry L.
Russell, Steven
author_sort Chan, Yuk-Sang
collection PubMed
description 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.
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spelling pubmed-37693822013-09-13 The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive Chan, Yuk-Sang Takeuchi, Ryo Jarjour, Jordan Huen, David S. Stoddard, Barry L. Russell, Steven PLoS One Research Article 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. Public Library of Science 2013-09-10 /pmc/articles/PMC3769382/ /pubmed/24040217 http://dx.doi.org/10.1371/journal.pone.0074254 Text en © 2013 Chan et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Chan, Yuk-Sang
Takeuchi, Ryo
Jarjour, Jordan
Huen, David S.
Stoddard, Barry L.
Russell, Steven
The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive
title The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive
title_full The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive
title_fullStr The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive
title_full_unstemmed The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive
title_short The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive
title_sort design and in vivo evaluation of engineered i-onui-based enzymes for heg gene drive
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769382/
https://www.ncbi.nlm.nih.gov/pubmed/24040217
http://dx.doi.org/10.1371/journal.pone.0074254
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