Figure 1.
Genome-wide CRISPR KO screen identified ESCRT machinery as a regulator of tauRD (P301L) clearance. (A) Schematic diagram of the genome-wide CRISPR KO screen. (B) β-score plot of all genes used in the CRISPR KO screen with the cutoff value of positive hits denoted as a red dashed line. 486 candidate genes were identified in the screen. Selected proteasome subunits, the proteasome accessory factor VCP, and the ESCRT-I complex subunit TSG101 are labeled. (C) Known and predicted protein–protein interactions among the 486 candidates identified in the CRISPR KO screen based on STRING analysis (ver11.5). (D) TauRD (P301L)-Venus aggregate–positive cells were transfected with control or individual sgRNA, as indicated, and successfully transfected cells were selected by puromycin treatment. TauRD (P301L)-Venus fluorescence was measured by flow cytometry 1 wk after transfection. Data were normalized to the Venus fluorescence of sgControl-treated cells and represent the mean ± SEM (n = 4, from four independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with *P < 0.05, ***P < 0.001. n.s., not significant. (E) TauRD (P301L)-Venus aggregate–positive cells were transfected with control, CHMP4A, CHMP4B, CHMP4C, or CHMP4A/B/C siRNA. TauRD (P301L)-Venus fluorescence was measured by flow cytometry 72 h after transfection. Data were normalized to the Venus fluorescence of siControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with **P < 0.01, ***P < 0.001. (F) TauRD (P301L)-Venus aggregate–positive cells were transfected with control, VPS4A, VPS4B, or VPS4A/B/siRNA and analyzed as in E. Data were normalized to the Venus fluorescence of siControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with *P < 0.05, ***P < 0.001. n.s., not significant. (G–K) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (Control) or mCherry-tagged sgRNA-resistant variants of TSG101 (G), UBAP1 (H), VPS28 (I), VPS37A (J), and PTPN23 (K) were transfected with individual sgRNA of ESCRT-I complex subunits and analyzed as in D. Data were normalized to the Venus fluorescence of sgControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test with *P < 0.05, **P < 0.01. n.s., not significant.

Genome-wide CRISPR KO screen identified ESCRT machinery as a regulator of tauRD (P301L) clearance. (A) Schematic diagram of the genome-wide CRISPR KO screen. (B) β-score plot of all genes used in the CRISPR KO screen with the cutoff value of positive hits denoted as a red dashed line. 486 candidate genes were identified in the screen. Selected proteasome subunits, the proteasome accessory factor VCP, and the ESCRT-I complex subunit TSG101 are labeled. (C) Known and predicted protein–protein interactions among the 486 candidates identified in the CRISPR KO screen based on STRING analysis (ver11.5). (D) TauRD (P301L)-Venus aggregate–positive cells were transfected with control or individual sgRNA, as indicated, and successfully transfected cells were selected by puromycin treatment. TauRD (P301L)-Venus fluorescence was measured by flow cytometry 1 wk after transfection. Data were normalized to the Venus fluorescence of sgControl-treated cells and represent the mean ± SEM (n = 4, from four independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with *P < 0.05, ***P < 0.001. n.s., not significant. (E) TauRD (P301L)-Venus aggregate–positive cells were transfected with control, CHMP4A, CHMP4B, CHMP4C, or CHMP4A/B/C siRNA. TauRD (P301L)-Venus fluorescence was measured by flow cytometry 72 h after transfection. Data were normalized to the Venus fluorescence of siControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with **P < 0.01, ***P < 0.001. (F) TauRD (P301L)-Venus aggregate–positive cells were transfected with control, VPS4A, VPS4B, or VPS4A/B/siRNA and analyzed as in E. Data were normalized to the Venus fluorescence of siControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Dunnett’s test with *P < 0.05, ***P < 0.001. n.s., not significant. (G–K) TauRD (P301L)-Venus aggregate–positive cells stably expressing mCherry (Control) or mCherry-tagged sgRNA-resistant variants of TSG101 (G), UBAP1 (H), VPS28 (I), VPS37A (J), and PTPN23 (K) were transfected with individual sgRNA of ESCRT-I complex subunits and analyzed as in D. Data were normalized to the Venus fluorescence of sgControl-treated cells and represent the mean ± SEM (n = 3, from three independent experiments). Significance was calculated using one-way ANOVA with Tukey’s test with *P < 0.05, **P < 0.01. n.s., not significant.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal