A silent intergenic locus for efficient and versatile transgene expression in Plasmodium falciparum
- 1Radboudumc
Abstract
Genetically modified Plasmodium falciparum parasites expressing transgenes such as fluorescent markers are crucial tools for malaria research. However, current reporter lines often rely on integrated transgene constructs that disrupt the coding sequence of a native gene, potentially affecting progression in the life cycle of the tool parasite line. We identified six candidate Silent Intergenic Loci (SILs): putatively editable intergenic regions without transcriptional activity, where gene insertions occurred in P. falciparum after its divergence from Plasmodium berghei and Plasmodium vivax. We characterised the largest candidate SIL on chromosome 12, SIL12, by integration of a constitutively expressed cytosolic mScarlet reporter. The resulting PfRed12 parasites progressed normally throughout the life cycle, from asexual blood-stage replication to mosquito stages and subsequent liver infection. Importantly, they infected both Anopheles stephensi and Anopheles coluzzii mosquitoes. PfRed12 parasites displayed bright fluorescence in both blood and mosquito stages, making them well suited for live visualisation of parasite transmission. In addition, we used two CRISPR-Cas9 target sites in the mScarlet cassette, one of which we purposely added, to perform efficient re-editing of the transgene. As a proof of concept, we re-edited PfRed12 parasites and integrated an apicoplast metabolic bypass construct, an inducible gametocyte producer construct, and two mNeonGreen reporter constructs. Together, our findings demonstrate that SIL12 permits stable transgene expression without compromising parasite development throughout the life cycle. We established PfRed12 as a new reporter line to study blood- and mosquito-stage development and as a versatile parental line for further genetic engineering.
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BibTeX
@misc{akkerman2026silent,
title = {{A silent intergenic locus for efficient and versatile transgene expression in Plasmodium falciparum}},
author = {L. J. Akkerman and S. Tassan-Lugrezin and E. T. Bekkering and F.-R. Lorenz and G.-J. van Gemert and N. I. Proellochs and T. W. A. Kooij and L. E. de Vries},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.13.758768},
url = {https://www.biorxiv.org/content/10.64898/2026.10.13.758768v1},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - A silent intergenic locus for efficient and versatile transgene expression in Plasmodium falciparum
AU - L. J. Akkerman
AU - S. Tassan-Lugrezin
AU - E. T. Bekkering
AU - F.-R. Lorenz
AU - G.-J. van Gemert
AU - N. I. Proellochs
AU - T. W. A. Kooij
AU - L. E. de Vries
PY - 2026
DA - 2026/10/13
PB - bioRxiv
DO - 10.64898/2026.10.13.758768
UR - https://www.biorxiv.org/content/10.64898/2026.10.13.758768v1
AB - Genetically modified Plasmodium falciparum parasites expressing transgenes such as fluorescent markers are crucial tools for malaria research. However, current reporter lines often rely on integrated transgene constructs that disrupt the coding sequence of a native gene, potentially affecting progression in the life cycle of the tool parasite line. We identified six candidate Silent Intergenic Loci (SILs): putatively editable intergenic regions without transcriptional activity, where gene insertions occurred in P. falciparum after its divergence from Plasmodium berghei and Plasmodium vivax. We characterised the largest candidate SIL on chromosome 12, SIL12, by integration of a constitutively expressed cytosolic mScarlet reporter. The resulting PfRed12 parasites progressed normally throughout the life cycle, from asexual blood-stage replication to mosquito stages and subsequent liver infection. Importantly, they infected both Anopheles stephensi and Anopheles coluzzii mosquitoes. PfRed12 parasites displayed bright fluorescence in both blood and mosquito stages, making them well suited for live visualisation of parasite transmission. In addition, we used two CRISPR-Cas9 target sites in the mScarlet cassette, one of which we purposely added, to perform efficient re-editing of the transgene. As a proof of concept, we re-edited PfRed12 parasites and integrated an apicoplast metabolic bypass construct, an inducible gametocyte producer construct, and two mNeonGreen reporter constructs. Together, our findings demonstrate that SIL12 permits stable transgene expression without compromising parasite development throughout the life cycle. We established PfRed12 as a new reporter line to study blood- and mosquito-stage development and as a versatile parental line for further genetic engineering.
ER -