title: 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.
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Catherin Marin
authors.initials: CM
authors.lastname: Mogollon
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Fiona J A
authors.initials: FJ
authors.lastname: van Pul
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Takashi
authors.initials: T
authors.lastname: Imai
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Jai
authors.initials: J
authors.lastname: Ramesar
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Séverine
authors.initials: S
authors.lastname: Chevalley-Maurel
authors.affiliation: Department of Hematology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Guido M
authors.initials: GM
authors.lastname: de Roo
authors.affiliation: Department of Hematology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Sabrina A J
authors.initials: SA
authors.lastname: Veld
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Kroeze
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Blandine M D
authors.initials: BM
authors.lastname: Franke-Fayard
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Janse
authors.affiliation: Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Shahid M
authors.identifier: http://orcid.org/0000-0002-0533-4670
authors.initials: SM
authors.lastname: Khan
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004351:Drug Resistance / Q000187:drug effects / Q000235:genetics*; D000072669:Gene Editing; D018503:Genome, Protozoan; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000235:genetics; D009154:Mutation; D010963:Plasmodium falciparum / Q000235:genetics*
chemical_list: D000962:Antimalarials; D006576:Heterocyclic Compounds, 4 or More Rings; D007546:Isoquinolines; C000597341:SJ733