Improvement of CRISPR/Cas9 system by transfecting Cas9-expressing Plasmodium berghei with linear donor template

Naoaki Shinzawa,Fumiya Hiyoshi,Shiroh Iwanaga,Masao Yuda,Daisuke Motooka,Tsubasa Nishi

doi:10.1038/s42003-020-01138-2

Naoaki Shinzawa, Fumiya Hiyoshi + Show 4 more

Open Access

https://doi.org/10.1038/s42003-020-01138-2

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Abstract

Malaria is caused by infection with Plasmodium parasites and is a major public health concern. The CRISPR/Cas9 system is a promising technology, but still has technical problems, such as low efficiency and unexpected recombination. Here, we solved these problems by transfecting Cas9-expressing parasites with linear donor templates. The use of a linear donor template prevented unexpected recombination; in addition, constitutive expression of Cas9 enabled immediate cleavage of the target locus after transfection, allowing efficient integration of the donor template. Furthermore, due to the absence of the cNHEJ pathway, there were no off-target mutations in the resultant parasites. In addition, this developed method could be applied for multiple genetic modifications on different chromosomes and for large-scale chromosomal deletion in the subtelomeric region. Because of its robustness, high efficiency, and versatile applicability, we hope this method will be standard in the post-genomic era of Plasmodium species.

Highlights

In this study, to further improve the CRISPR/Cas[9] system of the parasite, we integrated the Cas[9] gene into the genome of a rodent malaria parasite, P. berghei, and generated a transgenic parasite that constitutively expressed Cas[9]
The transgenic parasites in which those two cassettes were incorporated were selected by positive screening with pyrimethamine, and subsequently the hdhfr–yfcu cassette was removed through negative selection by screening with 5-fluorocytosine (5-FC) (Supplementary Fig. 1a)
Similar results were obtained using the same primer sets in biologically independent replicates, which were obtained by another transfection experiment. These results suggested that homology-directed repair (HDR) occurred between the donor template and the imc1i locus, while unexpected recombination might occur at the genomic locus of imc1i (Fig. 2a)

Summary

Introduction

To further improve the CRISPR/Cas[9] system of the parasite, we integrated the Cas[9] gene into the genome of a rodent malaria parasite, P. berghei, and generated a transgenic parasite that constitutively expressed Cas[9]. When we used the circular plasmid carrying both the donor template and the sgRNA as in the conventional CRISPR/Cas[9] system, an additional copy was unexpectedly incorporated at the target genomic locus by single crossover recombination, indicating that it was a serious overlooked technical limitation of the CRISPR/Cas[9] system to use plasmid DNA in the parasites. We solved this technical limitation by using a linear donor template and succeeded to engineer genes with high accuracy without any unexpected recombination. We further discuss the principle of genetic modification by the CRISPR/Cas[9] system in parasites and the possible application of the developed system

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Conclusion