Figure EV3. Insertion of CPSF3 ^E545K (AN3661‐resistant) in the SAG1 locus.

1. Schematic overview of the SAG1 gene editing strategy with CRISPR/Cas9 and the CPSF3 ^E545K resistance cassette. Wild‐type parasites (RH ku80) were transfected with the CRISPR/Cas9 vector, producing sgSAG1 RNA that targets Cas9 to the SAG1 coding sequence. After cleavage by Cas9, homology‐dependent repair was directed by a donor PCR amplicon encompassing CPSF3 ^E545K coding sequence with 5′ and 3′ regulatory sequences flanked by 60‐bp sequences homologous to SAG1. Transfected parasites were selected in the presence of 5 μM AN3661. Insertion of the CPSF3 ^E545K cassette within SAG1 was verified by PCR analysis in all clones using the indicated primers (in blue); bands at 6,500 bp indicate correct insertion of CPSF3 ^E545K, and the band at 370 bp is specific for wild‐type SAG1.
2. Detection of SAG1 by immunofluorescence. The parental wild‐type strain (RH ku80) and a SAG1 mutant strain [RH ku80 SAG1::CPSF3 ^E545K, clone #1 in (A)] were stained using anti‐SAG1 antibodies. Scale bars represent 10 μm.