Fig. 1

Redesigned marker-free CRISPR/Cas9-mediated deletion of the Pfset2 locus. a Construct used for Pfset2 gene disruption. Introns 1 to 4 of the Pfset2 locus are represented as gray boxes. pGFP-CBS-Pfset2 was designed to induce a double-strand break (DSB) near the 5’ end of intron 2. The Avi-tag between the homology arms was added to detect donor integration in the design of the PCR primers. Pf U6 5’, Pf U6 spliceosomal RNA promoter region; Pf CAM5’, Pf calmodulin promoter region; Pf Hsp86 5’, Pf heat shock protein 86 promoter region; AmpR, ampicillin resistance gene; ori, replication origin; ko, knockout. The positions and directions of the primers P1 to P4 are indicated by the small black arrows. b PCR analysis of the parasite populations obtained after transfection. WT, wild-type; SET2Δ, Pfset2 knockout; d31, d38, d47, and d61, days 31, 38, 47, and 61 after transfection, respectively; FACS: fluorescence-activated cell sorting. c Laser confocal microscopy of the parasites expressing the GFP protein. d DNA sequencing confirmed a 1.5-kb deletion in the Pfset2 gene. The top panel shows the partial nucleotide sequences of the left and right arms from the parental strain. The bottom panel shows the 48-bp DNA insert between the left and right arms