Generation of P. falciparum plasmid constructs for gene deletion.

Target genes were deleted using previously published Cas9-mediated gene editing methods (31). The homology repair plasmid pL8 (34) or pRS (31) was used in combination with the Cas9-expressing pUF1-Cas9 plasmid (34). Alternatively, genes were targeted for deletion using the pRSng (32) or pRSng (BSD) plasmid in combination with the pCasG plasmid (31). To generate the pRSng (BSD) plasmid, harmonized blasticidin-S-deaminase (hBSD) was cut with BamHI and HindIII from a synthetic plasmid (79) and ligated into the same sites in the pRSng plasmid to replace the sequence encoding human dihydrofolate reductase (hDHFR).

For generation of the deletion constructs, homology arms of ∼300 to 600 bp were amplified from P. falciparum strain NF54 genomic DNA (gDNA) using the homology arm 1 (HA1) and HA2 forward and reverse primers corresponding to each gene (Table S1) and inserted into the repair plasmids using ligation-independent cloning (LIC) methods (In-Fusion; Clontech). The repair plasmids were digested with NotI for insertion of HA1 and with NgoMIV for insertion of HA2. Guide RNA sequences were synthesized as oligonucleotides (Table S1), annealed, and inserted using LIC.