Abstract
Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.
Highlights
Malaria remains a major public health threat around the world
The plasmids used in our study were constructed based on the pL6cs-single guide RNA (sgRNA) and pUF1-Cas9 plasmids as described previously (Ghorbal et al, 2014; Figure 1A). pL6cs-sgRNA is the vector of sgRNA expression cassette and donor DNA, and expresses hDHFR marker for positive selection with WR99210 (Fidock and Wellems, 1997). pUF1-Cas9 expresses Streptococcus pyogenes Cas9 ortholog (SpCas9) endonuclease, fused with a 3xFLAG tag, two nuclear location sequences (NLS), and the resistance gene of blasticidin S deaminase (BSD)
The parental Cas9i line, in which the Cas9 expression cassette was integrated into genomic locus by a single-crossover recombination event (Collins et al, 2013), was generated to express stable Cas9 endonuclease, so that the pUF1-Cas9 plasmid is no longer needed for electroporation when using the CRISPR/Cas9 system (Figure 1B)
Summary
Malaria remains a major public health threat around the world. In 2018, it was estimated that approximately 228 million cases of malaria occurred worldwide (WHO, 2019). Among the five malarial species infecting humankind, Plasmodium falciparum accounts for the most severe malaria cases in African countries. Plasmodium parasites harbor a complex life cycle, which is subject to precisely spatiotemporal gene regulation (Philip and Waters, 2015). Since the genome sequencing of P. falciparum, the study of gene functions became a hot topic, with focus turned toward the underlying molecular mechanisms of immune evasion, pathogenesis, transmission, and drug. Optimized CRISPR/Cas System in P. falciparum resistance. To eliminate the global risk of malaria, basic research of the gene function and molecular mechanism of Plasmodium must be expanded
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