Highly efficient CRISPR/Cas9 system in Plasmodium falciparum using Cas9-expressing parasites and a linear donor template

Shiroh Iwanaga,Masao Yuda,Tsubasa Nishi,Naoaki Shinzawa

doi:10.1038/s41598-021-97984-z

Shiroh Iwanaga, Masao Yuda + Show 2 more

Open Access

https://doi.org/10.1038/s41598-021-97984-z

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Abstract

The CRISPR/Cas9 system is a powerful genetic engineering technology for Plasmodium falciparum. We here report further improvement of the CRISPR/Cas9 system by combining the Cas9-expressing parasite with a liner donor template DNA. The Cas9-expressing parasite was generated by inserting the cas9 gene in the genome by double crossover recombination. The site-directed mutagenesis and the fusion of fluorescence protein was achieved within two weeks with high efficiency (> 85%), by transfecting the schizonts of the Cas9-expressing parasite with the liner donor template and the plasmid carrying the sgRNAs. Notably, there were neither off-target mutations in the resultant transgenic parasites nor unexpected recombination, that are the technical problems of the current CRISPR/Cas9 system. Furthermore, with our system, two genes on different chromosomes were successfully modified in single transfection. Because of its high efficiency and robustness, our improved CRISPR/Cas9 system will become a standard technique for genetic engineering of P. falciparum, which dramatically advances future studies of this parasite.

Highlights

Cas9/sgRNA were directly introduced into purified schizonts, which are RBC-invasive form of parasites, and transgenic parasites were generated
Transfection method using the parasites at this developmental stage has been first developed in rodent malaria parasites, such as P. berghei[17], followed by P. knowlesi[18] and P. falciparum[2,15,19]
When 1.0 × 1 03–1.0 × 1 04 of rodent malaria parasite (P. berghei) were transfected independently, it was sufficient for genetic modification by the CRISPR/Cas[9] system using the Cas9-expressing parasite and the liner donor template[13,17]

Summary

Introduction

Cas9/sgRNA were directly introduced into purified schizonts, which are RBC-invasive form of parasites, and transgenic parasites were generated. Any unexpected recombination did not occur in the resultant transgenic parasites This suggests that to solve the technical problem and limitation associated with the current CRIPSR/ Cas[9] system, it might be important to use the Cas9-expressing parasite and a linear template DNA in combination. We were able to engineer two genes on different chromosomes simultaneously by using two linear donor templates and a plasmid with two sgRNAs, showing the applicability of our system This improved system solved the current technical problems of the CRISPR/Cas[9] system in P. falciparum and will be the standard method for genetic engineering of this parasite

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Conclusion