The malaria parasite has a complex, multistage life cycle occurring within two living beings, the vector mosquitoes and the vertebrate hosts. Its unique biology has fascinated many parasitologists. Also, as you know, malaria is one of the most major public health concerns, and it is important to elucidate the parasite molecular biology. Since the 1960s, biochemical and immunological studies have been energetically accomplished, and the molecular basis of the parasitism has become clear. Subsequently, the molecular biology has dramatically progressed by the development of a genetic modification method using a drug resistance cassette by Andrew P. Waters, Chris J. Janse et al. at Leiden University, which was published twenty-five years ago ¹. Gene targeting using drug marker has enabled gene knockout and knock-in of GFP or tag sequences, and it has become possible to perform molecular genetic analysis similar to model organisms. Recently, the genome-wide knockout analysis (PlasmoGEM) with this method created a phenotype database of individual genes, which means that the malaria genetics reached a certain goal ². It was a "revolution".

Since the development of the genetic modification method, functional analyses of various parasite genes have been carried out, greatly assisting the elucidation of the parasite life cycle. However, because the method using a single drug resistance cassette are limited to single gene manipulation, the biological pathways involving multiple factors remain largely unexplored. To fully understand them, multiple genetic manipulation based on the drug marker-free method are required. CRISPR/Cas9 was developed in malaria parasites in 2014 and expected as a drug marker-free method ^3,4. However, CRISPR/Cas9 in model organisms, such as yeast, fruit fly and rodents, were explosively spread but still has challenged in malaria research. The reason for this was considered to be that the current method requires two circular plasmids, thus making the procedure of drug selection more complex. In addition, the usage of the circular donor plasmid might result in the unexpected single crossover integration when non-essential genes were engineered.

We came up with two improvements to the CRISPR/Cas9 in malaria parasites. The first one was constitutive-expression of Cas9 nuclease to reduce the number of selectable drugs. The second one was the usage of linear form DNA as the donor DNA to avoid the single crossover recombination. Cas9 constitutive expressing line (named pbcas9) was transfected with the PCR-based linear donor DNA and the sgRNA expression plasmid to introduce point mutations. Surprisingly, the genotype of the parasites emerged after drug selection was all mutated and no wild type was detected, even before parasite cloning. Furthermore, no nucleotide insertions or deletions were detected at all, as the malaria parasite lacked the components of non-homologous end-joining. Taken together, the combination of three elements: the constitutive expression of Cas9, the use of linear donor DNA, and the absence of NHEJ mechanism created a perfect genetic modification method.

Because of its robustness, the pbcas9-based genetic modification was highly efficient not only for the allelic exchange, but also for other basic genetic modifications such as knockout by gene deletion and knock-in of fluorescent protein genes and tag sequences. In addition, we also succeeded in the modification of long genomic regions of >6 kb using two different sgRNAs and the large-scale chromosomal deletion with telomere seeding sequences. These techniques are truly genome editing and can be applicable to the analysis of chromosomal-level genome function. For details, please refer to our paper ⁵. We hope that the perfect genome editing developed in this study will once again lead to a breakthrough in the stagnant molecular biology of malaria parasites and create a new "revolution" for the first time in a quarter century.

References

1. van Dijk, M. R., Waters, A. P. & Janse, C. J. Stable transfection of malaria parasite blood stages. Science 268,1358–1362 (1995).
2. Schwach, F. et al. PlasmoGEM, a database supporting a community resource for large-scale experimental genetics in malaria parasites. Nucleic Acids Research 43,D1176–D1182 (2015).
3. Wagner, J. C., Platt, R. J., Goldfless, S. J., Zhang, F. & Niles, J. C. Efficient CRISPR-Cas9–mediated genome editing in Plasmodium falciparum. Nat Meth 11,915–918 (2014).
4. Ghorbal, M. et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol 32,819–821 (2014).
5. Shinzawa, N. et al. Improvement of CRISPR/Cas9 system by transfecting Cas9-expressing Plasmodium bergheiwith linear donor template. Communications Biology 1–13 (2020). doi:10.1038/s42003-020-01138-2