CRISPR-mediated Transfection of Brugia malayi.
The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was e...
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ftdoajarticles:oai:doaj.org/article:1c539a7ba01541c588b17e048ada3bc6 2023-05-15T15:13:18+02:00 CRISPR-mediated Transfection of Brugia malayi. Canhui Liu Alexandra Grote Elodie Ghedin Thomas R Unnasch 2020-08-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0008627 https://doaj.org/article/1c539a7ba01541c588b17e048ada3bc6 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0008627 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0008627 https://doaj.org/article/1c539a7ba01541c588b17e048ada3bc6 PLoS Neglected Tropical Diseases, Vol 14, Iss 8, p e0008627 (2020) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2020 ftdoajarticles https://doi.org/10.1371/journal.pntd.0008627 2022-12-31T08:01:35Z The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 14 8 e0008627 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Canhui Liu Alexandra Grote Elodie Ghedin Thomas R Unnasch CRISPR-mediated Transfection of Brugia malayi. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite. |
format |
Article in Journal/Newspaper |
author |
Canhui Liu Alexandra Grote Elodie Ghedin Thomas R Unnasch |
author_facet |
Canhui Liu Alexandra Grote Elodie Ghedin Thomas R Unnasch |
author_sort |
Canhui Liu |
title |
CRISPR-mediated Transfection of Brugia malayi. |
title_short |
CRISPR-mediated Transfection of Brugia malayi. |
title_full |
CRISPR-mediated Transfection of Brugia malayi. |
title_fullStr |
CRISPR-mediated Transfection of Brugia malayi. |
title_full_unstemmed |
CRISPR-mediated Transfection of Brugia malayi. |
title_sort |
crispr-mediated transfection of brugia malayi. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2020 |
url |
https://doi.org/10.1371/journal.pntd.0008627 https://doaj.org/article/1c539a7ba01541c588b17e048ada3bc6 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 14, Iss 8, p e0008627 (2020) |
op_relation |
https://doi.org/10.1371/journal.pntd.0008627 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0008627 https://doaj.org/article/1c539a7ba01541c588b17e048ada3bc6 |
op_doi |
https://doi.org/10.1371/journal.pntd.0008627 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
14 |
container_issue |
8 |
container_start_page |
e0008627 |
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1766343864992399360 |