Journal: Life Science Alliance
Article Title: CRISPRi screening reveals regulators of tau pathology shared between exosomal and vesicle-free tau
doi: 10.26508/lsa.202201689
Figure Lengend Snippet: (A) Schematic representation of the workflow for functional validation. Individual sgRNAs were used to silence the corresponding genes in combination with KRAB-dCas9 in tau biosensor cells (BSKRAB-KD), then treated with either sarkosyl-insoluble tau (vesicle-free tau seeds) or exosome-like EVs for 72 h, followed by detection and quantification of tau aggregation using FRET flow cytometry. A fraction of the same BSKRAB-KD cells was also grown for 72 h to corroborate the knockdown of protein expression using Western blots. (B, C, D, E, F, G) Integrated FRET intensities represent levels of tau aggregation upon knocking down the different targets. The control black column (NT) is the average obtained with three independent non-targeting sgRNAs (n = 3) assessed in triplicate. Control cells were compared with knockdown cells targeted individually (1, 2, and 3). Error bars represent the SEM for n = 3, * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001. Each single targeting sgRNA increased tau aggregation with both exosomal and vesicle-free tau seeds. (C, D) Interestingly, ANKLE2 and BANF1 (C, D) appear to induce a stronger effect on tau aggregation induced by exosome-like EVs. (H) Quantitative Western blot analysis of BSKRAB-KD knockdown cells. Each sgRNA generated a protein knockdown of the targeted gene. Note that the ANKLE2-specific antibody reacted with several isoforms, including the canonical variant sized 104–117 kD (red box outline); however, all isoforms were down-regulated when the ANKLE2 locus was silenced. Similarly, the EIF1AD antibody recognized the canonical isoform of 19 kD and one additional variant of lower molecular weight, both being silenced with the individual sgRNA against EIF1AD. (I) Quantification of the extent of protein knockdown for the different targeted genes. Error bars represent the SEM for n = 3, * P < 0.05; ** P < 0.01; and **** P < 0.0001.
Article Snippet: The following antibodies were used: anti-human ANKLE2 rabbit polyclonal (1:1,000; A302-965A; Thermo Fisher Scientific), anti-human EIF1AD rabbit polyclonal (1:1,000; 20528-1-AP; Thermo Fisher Scientific), anti-BANF1/BAF rabbit monoclonal (1:1,000; ab129184; Abcam), anti-human NUSAP1 rabbit polyclonal (1:500; ab137230; Abcam), anti-human VPS18 rabbit monoclonal (1:1,000; ab178689; Abcam), anti-human Tau-13 mouse monoclonal (1:1,000; MMS-520R; Covance), anti-tau phospho-Ser422 rabbit polyclonal (1:1,000; GTX86147; GeneTex), and the normalizers anti-GAPDH mouse monoclonal (1:2,000; MAB374; Millipore) and anti-GAPDH rabbit polyclonal (1:2,000; 10494-1-AP; Proteintech).
Techniques: Functional Assay, Biomarker Discovery, Flow Cytometry, Knockdown, Expressing, Western Blot, Control, Generated, Variant Assay, Molecular Weight
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