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Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9

BACKGROUND: Plasmodium falciparum is the deadliest malaria parasite. Currently, there are seldom commercial antibodies against P. falciparum proteins, which greatly limits the study on Plasmodium. CRISPR/Cas9 is an efficient genome editing method, which has been employed in various organisms. Howeve...

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Autores principales: Kuang, Dexuan, Qiao, Jichen, Li, Zhou, Wang, Weiwei, Xia, Hui, Jiang, Lubin, Dai, Jiejie, Fang, Qiang, Dai, Xueyu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712073/
https://www.ncbi.nlm.nih.gov/pubmed/29197418
http://dx.doi.org/10.1186/s13071-017-2539-0
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author Kuang, Dexuan
Qiao, Jichen
Li, Zhou
Wang, Weiwei
Xia, Hui
Jiang, Lubin
Dai, Jiejie
Fang, Qiang
Dai, Xueyu
author_facet Kuang, Dexuan
Qiao, Jichen
Li, Zhou
Wang, Weiwei
Xia, Hui
Jiang, Lubin
Dai, Jiejie
Fang, Qiang
Dai, Xueyu
author_sort Kuang, Dexuan
collection PubMed
description BACKGROUND: Plasmodium falciparum is the deadliest malaria parasite. Currently, there are seldom commercial antibodies against P. falciparum proteins, which greatly limits the study on Plasmodium. CRISPR/Cas9 is an efficient genome editing method, which has been employed in various organisms. However, the use of this technique in P. falciparum is still limited to gene knockout, site-specific mutation and generation of green fluorescent protein (GFP) reporter line with disruption of inserted sites. RESULTS: We have adapted the CRISPR/Cas9 system to add commercial tag sequences to endogenous genes of P. falciparum. To add HA or HA-TY1 tags to ck2β1, ck2α and stk, pL6cs-hDHFR-ck2β1/ck2α/stk was constructed, which contained sequences of tags, specific homologous arms, and sgRNA. The P. falciparum 3D7 strain was subsequently transfected with pUF1-BSD-Cas9 and pL6cs-hDHFR-ck2β1/ck2α/stk plasmids via electroporation. After that, BSD and WR99210 drugs were added to the culture to screen parasites containing both plasmids. Twenty days after electroporation, live parasites appeared and were collected to check the tagging by PCR, DNA sequencing, Western blotting and immuno-fluorescence assays. The results showed that the tags were successfully integrated into the C-terminus of these three proteins. CONCLUSIONS: We have improved the method to integrate tags to Plasmodium falciparum genes using the CRISPR/Cas9 method, which lays the foundation for further study of Plasmodium falciparum at the molecular level. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13071-017-2539-0) contains supplementary material, which is available to authorized users.
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spelling pubmed-57120732017-12-06 Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9 Kuang, Dexuan Qiao, Jichen Li, Zhou Wang, Weiwei Xia, Hui Jiang, Lubin Dai, Jiejie Fang, Qiang Dai, Xueyu Parasit Vectors Research BACKGROUND: Plasmodium falciparum is the deadliest malaria parasite. Currently, there are seldom commercial antibodies against P. falciparum proteins, which greatly limits the study on Plasmodium. CRISPR/Cas9 is an efficient genome editing method, which has been employed in various organisms. However, the use of this technique in P. falciparum is still limited to gene knockout, site-specific mutation and generation of green fluorescent protein (GFP) reporter line with disruption of inserted sites. RESULTS: We have adapted the CRISPR/Cas9 system to add commercial tag sequences to endogenous genes of P. falciparum. To add HA or HA-TY1 tags to ck2β1, ck2α and stk, pL6cs-hDHFR-ck2β1/ck2α/stk was constructed, which contained sequences of tags, specific homologous arms, and sgRNA. The P. falciparum 3D7 strain was subsequently transfected with pUF1-BSD-Cas9 and pL6cs-hDHFR-ck2β1/ck2α/stk plasmids via electroporation. After that, BSD and WR99210 drugs were added to the culture to screen parasites containing both plasmids. Twenty days after electroporation, live parasites appeared and were collected to check the tagging by PCR, DNA sequencing, Western blotting and immuno-fluorescence assays. The results showed that the tags were successfully integrated into the C-terminus of these three proteins. CONCLUSIONS: We have improved the method to integrate tags to Plasmodium falciparum genes using the CRISPR/Cas9 method, which lays the foundation for further study of Plasmodium falciparum at the molecular level. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13071-017-2539-0) contains supplementary material, which is available to authorized users. BioMed Central 2017-12-02 /pmc/articles/PMC5712073/ /pubmed/29197418 http://dx.doi.org/10.1186/s13071-017-2539-0 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Kuang, Dexuan
Qiao, Jichen
Li, Zhou
Wang, Weiwei
Xia, Hui
Jiang, Lubin
Dai, Jiejie
Fang, Qiang
Dai, Xueyu
Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9
title Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9
title_full Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9
title_fullStr Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9
title_full_unstemmed Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9
title_short Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9
title_sort tagging to endogenous genes of plasmodium falciparum using crispr/cas9
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712073/
https://www.ncbi.nlm.nih.gov/pubmed/29197418
http://dx.doi.org/10.1186/s13071-017-2539-0
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