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. 2017 Jun 20;7:3881. doi: 10.1038/s41598-017-03984-3

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Successful generation of P. falciparum parasite lines expressing DiCre recombinase using CRISPR-Cas9. (a) Schematic of wild type p230p ORF and modified ORF containing the DiCre recombinase expression cassettes following transfection of 3D7 with pBSp230pDiCre and pDC120 (pDC2-hDHFR-yFCU plasmid carrying the guide sequence). Homology regions (HRs) used for targeting were 500 bp (HR1) and 644 bp (HR2) in size. (b) Schematic of wild type pfs47 locus and modified, DiCre-expressing pfs47 locus. The blue box depicts the pfs47 ORF, and hashed boxes the DNA regions used for homologous repair. Transfection of 3D7 with pBSPfs47DiCre and pDC287 (the plasmid carrying the guide sequence for Pfs47 targeting) resulted in the disruption of the pfs47 ORF. (c) PCR screen on wild type and transgenic parasite clones (II-3 and Pfs47-13) showing successful integration of the DiCre cassette into the targeted loci. Primer pairs 1/2 and 3/4 amplify fragments of p230p in wild type parasites only with expected sizes of 772 bp and 771 bp respectively. Primer pairs 1/5 and 4/6 amplify DNA only after successful integration into p230p with expected sizes of 947 bp and 1229 bp respectively. Primer pairs 7/8 and 9/10 amplify 1042 bp and 1114 bp fragments from the pfs47 wild type locus only; whereas primer pairs 5/7 and 6/10 only amplify the expected 1257 bp and 1555 bp bands after successful integration. Primers used for amplification are shown on the top of each panel, as well as in the schematics in a) and b). DNA marker sizes are indicated on the left in kb.