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 fli...
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ftdoajarticles:oai:doaj.org/article:51be2b5f5ffb4fe0a30f2569284c2437 2023-05-15T15:15:57+02:00 Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9. Ines Martin-Martin Azadeh Aryan Claudio Meneses Zach N Adelman Eric Calvo 2018-09-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0006769 https://doaj.org/article/51be2b5f5ffb4fe0a30f2569284c2437 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC6150542?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0006769 https://doaj.org/article/51be2b5f5ffb4fe0a30f2569284c2437 PLoS Neglected Tropical Diseases, Vol 12, Iss 9, p e0006769 (2018) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2018 ftdoajarticles https://doi.org/10.1371/journal.pntd.0006769 2022-12-31T05:44:47Z 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. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 12 9 e0006769 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Ines Martin-Martin Azadeh Aryan Claudio Meneses Zach N Adelman Eric Calvo Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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 |
Article in Journal/Newspaper |
author |
Ines Martin-Martin Azadeh Aryan Claudio Meneses Zach N Adelman Eric Calvo |
author_facet |
Ines Martin-Martin Azadeh Aryan Claudio Meneses Zach N Adelman Eric Calvo |
author_sort |
Ines Martin-Martin |
title |
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_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_sort |
optimization of sand fly embryo microinjection for gene editing by crispr/cas9. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2018 |
url |
https://doi.org/10.1371/journal.pntd.0006769 https://doaj.org/article/51be2b5f5ffb4fe0a30f2569284c2437 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 12, Iss 9, p e0006769 (2018) |
op_relation |
http://europepmc.org/articles/PMC6150542?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0006769 https://doaj.org/article/51be2b5f5ffb4fe0a30f2569284c2437 |
op_doi |
https://doi.org/10.1371/journal.pntd.0006769 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
12 |
container_issue |
9 |
container_start_page |
e0006769 |
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1766346281098149888 |