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Fig. 2 | Malaria Journal

Fig. 2

From: Improved Plasmodium falciparum dilution cloning through efficient quantification of parasite numbers and c-SNARF detection

Fig. 2

Plasmid constructs used in transfections. In A, the proposed model for single crossover recombination of the plasmid gene-GFP-HA is shown. The HR (homology region) consists of a sequence of 1013 bp, 76% A/T and the transfectant was selected using an hDHFR cassette. In B, the proposed model for a CRISPR/Cas9 transfection for insertion of a LoxP and a HA tag sequence into the target open reading frame (ORF). The transfection was performed into a parasite line already expressing Cas9 and the selection was done using blasticidin (2.5 µg/ml). HR1 (homolog region 1) consists of a target region in the coding region of the target gene (about 270 bp with 72% A/T) and HR2 target localizes in the 3’ untranslated region (3′ UTR); the size of HR2 was about 280 bp with 87% A/T. The red dashed line represents the NGG PAM site where Cas9 cleaves, mediated by the specific sgRNA. The yellow box represents the recodonized sequence which substitutes the original sgRNA target sequence to prevent continued cleavage of Cas9

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