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. 2023 Jan 26;14:428. doi: 10.1038/s41467-023-36145-4

Fig. 1. CRISPR/Cas9 allows manipulation of A. castellanii.

Fig. 1

a Constructs used to constitutively express Cas9 in A. castellanii. The sequence of the gRNA is depicted with diamonds followed by the Cas9-binding scaffold and an A. castellanii tRNA (yellow rectangles). The localization of the GFP-Cas9 fusion for the different constructs is shown by fluorescence. Micrographs are representative of 3 independent experiments. Scale bar: 10μm. b Representative micrographs showing amoebas expressing mRFP (vector Vc241) and GFP-Cas9 after selection for 2–3 weeks with the appropriate drug(s) (refer to materials and methods). mRFP (product of the targeted gene) and GFP (Indicating Cas9 expression) fluorescence are shown. MCR: mutagenic chain reaction. Scale bar: 10μm. c The quantification of the micrograph shown in (b). The mean ± SD of at least 200 amoebas (3 independent experiments (n = 3)) is shown. Amoebas were classified either as non-fluorescent, mRFP+, GFP+ or GFP+mRFP+. MCR: mutagenic chain reaction. Guides targeting pandoravirus rpb1 were used as off-target gRNAs. Nd: not detected. d Representative sequencing results of targeted guide sequences on rfp upon transfection with on-target gRNAs. PCR were performed on the target sequences as shown in Fig. S1a, b, cloned into a TA cloning vector and single clones were sent for sequencing. The wild type sequence and mutations generated are shown in red and blue respectively. Ten individual clones were amplified and sequenced. e Schematic representation of the rfp locus, guide targeting location, homology arms for recombination and primer annealing sites for a disruption using a “mutagenic chain reaction” strategy (see Fig. S1e). f Gene disruption of rfp by the “mutagenic chain reaction” observed at the population level after 2–3 weeks post-transfection. Expected PCR size: a + b: 837 bp (unmodified locus), a + b 890 bp (recombinant locus), a + c: 500 bp (unmodified locus). Note that the primer b anneals both in the wild type and recombinant locus resulting in PCR products with slight differences in size.