Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol

Abstract

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.

Rapid and iterative genome editing in the zoonotic malaria parasitePlasmodium knowlesi: New tools forP. vivaxresearch

“…Effective CRISPR-Cas9 genome editing of malaria parasites therefore requires expression cassettes for the guide RNA and the Cas9 nuclease, and a DSB repair template (donor DNA) containing the desired change, flanked by two regions of homology to the genomic target. Whilst a variety of approaches have been used in P. falciparum , many embed these elements into two plasmids, each expressing a different drug-selectable marker (22, 27-30). This allows for selection of very rare events, but complicates construct design and is not ideal for multiple modifications of a given line – as both selectable markers must then be recycled.…”

Tagging to endogenous genes of Plasmodium falciparum using CRISPR/Cas9

“…In Plasmodium , the double-strand break repair by homologous recombination is largely favored since canonical NHEJ is deficient in this organism [ 20 ]. Up to now, the application of CRISPR/Cas9 system in P. falciparum was limited to gene knock out, generating genome mutation or GFP reporter parasite line with disruption of inserted sites [ 17 , 19 , 21 ]. A recently improved CRISPR/Cas9 system contains Cas9 nuclease, sgRNA, and a selectable marker in one plasmid while homologous arms (donor DNA fragments) without selectable marker in another plasmid.…”

“…The DSBs are then repaired by homologous recombination using donor DNAs since the canonical nonhomologous end-joining (NHEJ) is deficient in Plasmodium [ 20 ]. This technique has already been used in P. falciparum for gene knock out, generating single-nucleotide substitutions and a green fluorescent protein (GFP) reporter line with disruption of inserted sites [ 17 , 19 , 21 ], but the adaption of this system for adding tags to P. falciparum genes has not been reported yet.…”

Exposure to artemisinin at the trophozoite stage increases sexual conversion rates in the malaria parasite Plasmodium falciparum

“…Cultures for the production of mature gametocytes for mosquito infection were maintained in a semi-automated shaker incubator system as described (Mogollon et al, 2016). Fresh human serum and red blood cells (RBC) for these experiments were obtained from the Dutch National Blood Bank (Sanquin Amsterdam, the Netherlands; 430 permission granted from donors for the use of blood products for malaria research and microbiology; tested for safety).…”