Cargando…

Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand fl...

Descripción completa

Detalles Bibliográficos
Autores principales: Martin-Martin, Ines, Aryan, Azadeh, Meneses, Claudio, Adelman, Zach N., Calvo, Eric
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150542/
https://www.ncbi.nlm.nih.gov/pubmed/30180160
http://dx.doi.org/10.1371/journal.pntd.0006769
_version_ 1783357008698671104
author Martin-Martin, Ines
Aryan, Azadeh
Meneses, Claudio
Adelman, Zach N.
Calvo, Eric
author_facet Martin-Martin, Ines
Aryan, Azadeh
Meneses, Claudio
Adelman, Zach N.
Calvo, Eric
author_sort Martin-Martin, Ines
collection PubMed
description BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed. CONCLUSION AND SIGNIFICANCE: The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.
format Online
Article
Text
id pubmed-6150542
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-61505422018-10-19 Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9 Martin-Martin, Ines Aryan, Azadeh Meneses, Claudio Adelman, Zach N. Calvo, Eric PLoS Negl Trop Dis Research Article BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed. CONCLUSION AND SIGNIFICANCE: The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions. Public Library of Science 2018-09-04 /pmc/articles/PMC6150542/ /pubmed/30180160 http://dx.doi.org/10.1371/journal.pntd.0006769 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication.
spellingShingle Research Article
Martin-Martin, Ines
Aryan, Azadeh
Meneses, Claudio
Adelman, Zach N.
Calvo, Eric
Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
title Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
title_full Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
title_fullStr Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
title_full_unstemmed Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
title_short Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9
title_sort optimization of sand fly embryo microinjection for gene editing by crispr/cas9
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150542/
https://www.ncbi.nlm.nih.gov/pubmed/30180160
http://dx.doi.org/10.1371/journal.pntd.0006769
work_keys_str_mv AT martinmartinines optimizationofsandflyembryomicroinjectionforgeneeditingbycrisprcas9
AT aryanazadeh optimizationofsandflyembryomicroinjectionforgeneeditingbycrisprcas9
AT menesesclaudio optimizationofsandflyembryomicroinjectionforgeneeditingbycrisprcas9
AT adelmanzachn optimizationofsandflyembryomicroinjectionforgeneeditingbycrisprcas9
AT calvoeric optimizationofsandflyembryomicroinjectionforgeneeditingbycrisprcas9