Plasmid-free CRISPR/Cas9 genome editing in Plasmodium falciparum confirms mutations conferring resistance to the dihydroisoquinolone clinical candidate SJ733
Fig 2
Sanger and NGS sequencing coverage of targeted CRISPR mutations at the pfatp4 locus for ACP-B6-L350H and ACP-B6-P412T with clonal wild type parent strain ACP-B6. Red bars delineate the respective 20 nt guide RNA target sites and PAM sites required for each edit. NGS coverage at each location is indicated by blue columns. (a) Sequencing data of targeted locus 1002–1072 in pfatp4 from strain ACP-B6-L350H showing SJ733 resistance-conferring SNPs in L350 and four other synonymous mutations introduced by CRISPR. Sequences of wild type pfatp4 and repair template ssODN L350H are shown in alignment. The two silent mutations in ssODN L350H located 39 and 42 nt away were not incorporated into ACP-B6-L350H. (b) Sequencing data of targeted locus 1206–1276 in pfatp4 from strain ACP-B6-P412T showing the SJ733 resistance-conferring SNP and silent mutations introduced by CRISPR. Sequences of wild type pfatp4 and repair template ssODN P412T are shown in alignment.