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bioRxiv · 10.64898/2026.11.09.765308

Engineering a High-Fidelity CRISPR-Cas13d System for Targeted RNA Depletion in Plasmodium falciparum

Serrano-Duran, R.·Lopez Farfan, D. C.·Moreno-Mateos, M. A.·Gomez-Diaz, E.
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Abstract

Functional genomics in Plasmodium falciparum is severely constrained by the evolutionary absence of an endogenous RNA interference pathway and the requirement for time-consuming direct target locus modification by conventional conditional genetic engineering systems. To overcome these limitations, we established a programmable post-transcriptional gene silencing system in P. falciparum using High-Fidelity RfxCas13d (HF-RfxCas13d), an RNA-guided RNA nuclease that has been previously tested in several eukaryotic hosts. Using CRISPR-SpyCas9 we integrated a HF-RfxCas13d expression cassette at the dispensable pfs47 locus and generated a stable marker-free transgenic line capable of constitutive HF-RfxCas13d cytosolic expression without impairing intraerythrocytic asexual growth. Episomal delivery of single or dual guide RNAs (gRNAs) achieved significant target mRNA depletion (41%-76%) across three endogenous loci atp4, ap2-exp, ap2-g genes. Applying this system to the master transcriptional regulator of gametocytogenesis (ap2-g) resulted in transcript depletion that functionally impaired sexual differentiation in committed parasites, reducing significantly the Sexual Conversion Rate a 70%. Overall, our novel HF-Cas13d line provides a flexible, scalable, and non-locus-modifying addition to the P. falciparum genetic toolbox to accelerate functional genomics and malaria drug target identification.

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Serrano-Duran, R., Lopez Farfan, D. C., Moreno-Mateos, M. A., Gomez-Diaz, E.. 2026-11-12. Engineering a High-Fidelity CRISPR-Cas13d System for Targeted RNA Depletion in Plasmodium falciparum. https://doi.org/10.64898/2026.11.09.765308

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