Journal: bioRxiv
Article Title: A phenotypic screen using splitCas9 identifies essential genes required for actin regulation during host cell egress and invasion by Toxoplasma gondii
doi: 10.1101/2021.09.24.461619
Figure Lengend Snippet: a, Schematic of the splitCas9-system. Transgenic parasites are generated co-expressing the two splitCas9 subunits together with a single-guide RNA (sgRNA). Upon addition of rapamycin the two subunits dimerise, leading to reconstituted Cas9 activity and therefore to disruption of the gene targeted by the sgRNA. b, Analysis of RHsCas9-gap40 parasites that were treated with 50 nM rapamycin for 48h before fixation using indicated antibodies. Scale bars are 5μm. Three distinct phenotypes can be observed: the gap40 phenotype, as described previously with collapsed IMC and loss of GAP40 expression (top panel, asterisk); a phenotype where some parasites within the PV are aberrant (bottom panel); and parasites with normal IMC and GAP40 localisation (top panel, arrow). c, Quantification of gap40 phenotypes shown in (a) in indicated parasite strains. Parasites were induced with or without rapamycin for the indicated time and fixed 48 h post infection. Only parasites expressing both the gap40sgRNA and the sCas9 system presented a gap40 phenotype after induction. Parasites were treated with rapamycin for 1h or the whole growth period of 48h as indicated. Data represents three independent experiments. For each condition 100 vacuoles were counted (total n=300). Average and standard deviation (SD) are represented. d, Analysis of RHsCas9-sag1 parasites that were treated with 50 nM rapamycin for 48h before fixation using indicated antibodies. Nuclei were stained with DAPI. Scale bars are 5μm. Three distinct phenotypes can be observed: healthy vacuoles lacking SAG1 (bottom panel, arrow); and parasites lacking SAG1 while displaying aberrant nuclear and cellular morphology (bottom panel, asterisk). e, Quantification of the phenotypes shown in (d). Aberrant nuclei and cellular morphology were observed only when sag1 was disrupted (KO) by sCas9 activation (1 st lytic cycle). Abundance of non-healthy parasites was reduced to background levels when induced RHsCas9- sag1 parasites were mechanically lysed, transferred onto fresh host cells and grown again for 48h in a second lytic cycle (2 nd generation; total incubation of 96h). Parasites were treated with rapamycin for 1h or for the whole growth period of 48h as indicated. Data represent three independent experiments. For each condition, 100 vacuoles were counted (total n=300). Average and standard deviation (SD) are represented. f, Analysis of indicator parasites expressing sgRNA targeting sag1. Disruption of sag1 has no effect on the F-actin network. Scale bars are 5 μm. g, Analysis of indicator parasites expressing indicated sgRNAs. IFA depicting the effect of drpA (RHsCas9- drpA ), or adf (RHsCas9- adf ), act1 (RHsCas9- act1) and frm2 (RHsCas9- frm2 ) disruption on actin network and apicoplast segregation. To achieve gene disruption (KO), parasites were incubated with 50 nM rapamycin for 1h and fixed after 48h.Nuclei were stained with DAPI. Scale bars are 5 μm. h, Depiction of indicator parasites co-expressing CbEm, FNR-RFP and the sCas9-subunits. Scale bar is 5 μm.
Article Snippet: The resulting plasmids were confirmed by sequencing. pU6- sag1 gRNA-scaffold and pU6-gap40 gRNA-scaffold sequence was synthesised and cloned into a backbone vector containing the DHFR resistance cassette by GeneScript.
Techniques: Transgenic Assay, Generated, Expressing, Activity Assay, Disruption, Infection, Standard Deviation, Staining, Activation Assay, Incubation
