Open AccessRapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol
Author(s) -
Catherin Marin Mogollon,
Fiona J. A. van Pul,
Takashi Imai,
Jai Ramesar,
Séverine Chevalley-Maurel,
Guido M. de Roo,
Sabrina A.J. Veld,
Hans Kroeze,
Blandine Franke-Fayard,
Chris J. Janse,
Shahid M. Khan
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0168362
Subject(s) - crispr , biology , green fluorescent protein , selectable marker , genome editing , cas9 , gene , transgene , genome , reporter gene , microbiology and biotechnology , genetics , gene expression
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.