Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis
CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-09-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2022.981432/full |
| _version_ | 1811261518949384192 |
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| author | Johanna Hoshizaki Hannah Jagoe Marcus C. S. Lee |
| author_facet | Johanna Hoshizaki Hannah Jagoe Marcus C. S. Lee |
| author_sort | Johanna Hoshizaki |
| collection | DOAJ |
| description | CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an in-frame blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation. |
| first_indexed | 2024-04-12T19:06:08Z |
| format | Article |
| id | doaj.art-b874966ba7fa4effaa1009cd37ee3274 |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-04-12T19:06:08Z |
| publishDate | 2022-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-b874966ba7fa4effaa1009cd37ee32742022-12-22T03:20:01ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882022-09-011210.3389/fcimb.2022.981432981432Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysisJohanna HoshizakiHannah JagoeMarcus C. S. LeeCRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an in-frame blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation.https://www.frontiersin.org/articles/10.3389/fcimb.2022.981432/fullmalariareporter genesfluorescenceCRISPR/Cas9Plasmodium falciparum |
| spellingShingle | Johanna Hoshizaki Hannah Jagoe Marcus C. S. Lee Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis Frontiers in Cellular and Infection Microbiology malaria reporter genes fluorescence CRISPR/Cas9 Plasmodium falciparum |
| title | Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis |
| title_full | Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis |
| title_fullStr | Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis |
| title_full_unstemmed | Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis |
| title_short | Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis |
| title_sort | efficient generation of mneongreen plasmodium falciparum reporter lines enables quantitative fitness analysis |
| topic | malaria reporter genes fluorescence CRISPR/Cas9 Plasmodium falciparum |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2022.981432/full |
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