S2 Fig -

<p><b>(A)</b> Schematic representation of the constructs used for <i>in situ</i> tagging of GiMlf with the BAP tag using the CRISPR/Cas9 system. In this case, the <i>pac</i> cassette, which provides resistance to puromycin is not part of the repair template. <b>(B)</b> Left—PCR amplification of the integrated BAP tag in the genomes of subcloned populations C11, D6, and E7 of <i>Giardia</i> modified by CRISPR/Cas9. The Cas9 cell line served as the parental cell line, NC-negative control. Right–PCR amplification of the genomic region containing the <i>gimlf</i> gene in modified and control lineages. One of the amplification primers was outside the recombination regions used for the integration of the BAP tag. Only one band was present in all the tested subclones, showing that the BAP tag was integrated into all genomic copies of <i>gimlf</i> in the modified lineages. <b>(C)</b> Western blot of whole cell lysates of the subcloned endogenously BAP-tagged GiMlf cell line (C11) and a control cell line detecting the expression of Cas9-HA (top panel) with anti-HA antibody, the shift in size and the presence of only a single double band when detected with anti-GiMlf polyclonal antibody (middle panel), and the presence of the BAP tag using anti-BAP antibody.</p> <p>(PDF)</p>