Divergent roles for the RH5 complex components, CyRPA and RIPR in human-infective malaria parasites
Fig 2
RIPR and CyRPA are essential for P. knowlesi parasite growth.
Schematics of disruption approaches for ripr (A) and cyrpa (C) in P. knowlesi, respectively. ORFs are depicted, indicating regions used as homology regions (HR) in repair plasmids. Black boxes represent endogenous DNA sequence (bp 1006–1054 (ripr) and 372–408 (cyrpa)) which was replaced either by recodonized DNA or by a triple HA-tag followed by a stop codon. Sequence details are given in the corresponding boxes below, of endogenous (wild type), recodonized DNA or HA-STOP regions. Primers used in PCR to genotype transfectants are indicated by arrows. B) Genotyping of PkA1-H.1 transfections with pGEMT-PkRIPRko or pGEMT-PkRIPRrecodonized with pDC596 and pDC645 Cas9/guide plasmids. D) Genotyping of PkA1-H.1 transfections using pGEMT-PkCyRPAko or pGEMT-PkCyRPArecodonized together with Cas9/guide plasmids pDC2915 or pDC2919. Primers for genotyping (S2 Table) were as follows: a, PKRIPRkoextFor; b, PKRIPRintRev; c, PKRIPRintFor; d, PKRIPRkoextRev; e, PKRIPRkorecRev; f, PKRIPRkorecodFor; g, HArev(Xma); h, HAfor(Xma); i, PKCyRPAextFor; j, PKCyRPAintRev; k, PKCyRPAintFor; l, PKCyRPAextRev; m, PKCyRPArecodRev; n, PKCyRPArecodFor. Expected PCR product sizes for Pkripr are: a/b = 426 bp; a/e = 434 bp; c/d = 412 bp; f/d = 418 bp; a/h = 509 bp; g/d = 499 bp. Expected PCR product sizes for Pkcyrpa are: i/j = 425 bp; i/m = 438 bp; k/l = 566 bp; n/l = 570 bp; i/h = 504 bp; g/l = 654 bp.