Fig. 3: CRISPR/Cas9 allows manipulation of nuclear GVs.

a Schematic depiction of genetic manipulation of GVs. A. castellanii expressing a nuclear or cytoplasmic Cas9 are shown in the left and right, respectively. VF: viral factory. N: Nucleus. b Quantification of the number (#) of viruses produced 24 h post-infection (hpi) of CRISPR/Cas9 expressing amoebas. Values are expressed relative to # of viral particles produced within amoebas expressing off-target gRNAs. A cytoplasmic version of Cas9 was used to attempt gene disruption of cytoplasmic GV (Mimivirus reunion²⁸ and Pithovirus sibericum²⁷) and a nuclear version of Cas9 was used for the gene disruption of nuclear GVs (Pandoravirus neocaledonia³ and Mollivirus kamchatka²⁶). Data correspond to the mean ± SD of 3 independent experiments. MOI = 1. Guides targeting mimivirus rpb1 were used as off-target gRNAs for pithovirus infection and vice versa. Guides targeting pandoravirus rpb1 were used as off-target gRNAs for the infection of mollivirus and vice versa. The null hypothesis (α  =  0.05) was tested using unpaired two-tailed Student’s t tests. c Multiple gRNA combinations were used to target the rpb1 gene of Pandoravirus neocaledonia. Data were performed as Fig. 2b. Guides targeting mollivirus mcp were used as off-target gRNAs. Sequence of the gRNA 1 to 6 are shown in Table S1. The null hypothesis (α  =  0.05) was tested using unpaired two-tailed Student’s t tests. d Schematic representation of the vector and strategy utilized for rbp2 endogenous tagging in Pandoravirus neocaledonia. Selection cassette was introduced by homologous recombination. e Cartoon depicting the strategy for selection of recombinant viruses. Viral infection was performed 1 h post-transfection. Ntc: Nourseothricin. P = passage. f Nuclear localization of endogenously tagged RPB2-FLAG shown by immunofluorescence assay using anti-FLAG antibodies. DAPI: nuclear marker. The immunofluorescence signal disappears upon infection of cells encoding CRISPR/Cas9 targeting the rpb2 locus. Guides targeting mollivirus mcp were used as off-target gRNAs. Scale bar: 10μm. g The quantification of the micrograph shown in (f). The mean ± SD of at least 200 amoebas (3 independent experiments (n = 3)) is shown. The null hypothesis (α  =  0.05) was tested using unpaired two-tailed Student’s t tests. Source data are provided as a Source Data file.