Genome-wide CRISPR-Cas9 KO screen using Pulse-Shape Analysis uncovers expected and novel modifiers of cellular proteostasis.

A) Schematic of theory underlying PulSA. B) Schematic of TDP-43 ΔNLS expression vectors tagged C- or N- terminally with mClover3, PulSA plots generated after transfection of corresponding vectors in 293Ts, and inset of cells showing TDP-43 localization. C) Schematic of genome-wide CRISPR-Cas9 KO screening method. D) Volcano plot of CRISPR screen results where each dot is the average of 4 sgRNAs targeting each gene, displaying the phenotype (x-axis) vs the significance (y-axis). Colored dots correspond to STRING cluster in 1F. E) Arrayed screen validation of top modifiers of TDP-43 ΔNLS aggregation. Each sgRNA KO is normalized to AAV5 non-targeting control and plotted to show the normalized change in the % of cells harboring TDP-43 ΔNLS aggregates. n = 3 replicates, one way anova with Tukey HSD test comparing AAV control to all 6 sgRNAs targeting each gene. Adjusted p -values: AAV-UBE3C 0.00038; AAV-HSPA4 0.03312; AAV-BTNL9 0.03011; AAV-SRRD 0.00003. F) Select clusters of top protein-protein interactions (STRING database) of top 119 genes ranked by p-value that when knocked out increase the number of cells harboring TDP-43 ΔNLS aggregates. Clusters generated with MCL clustering and excludes genes with no known connections and clusters with insignificant p-values. Clusters colored based on STRING annotated GO terms. Dashed line indicates noteworthy link of UBE3C to proteasomal degradation pathway G) Histograms of total TDP-43 ΔNLS expression for AAV5 non-targeting control and two HSPA4 targeting sgRNAs. F) Fraction of cells with TDP-43 aggregates (y-axis) in each TDP-43 ΔNLS expression bin (x-axis) for AAV5 non-targeting control and two HSPA4 targeting sgRNAs. Adjusted p-values i)bin 2500 AAV:HSPA4_C11 = 0.04711; AAV:HSPA4_C12 = 0.04054 ii) bin 3500 AAV:HSPA4_C11 = 0.03956; AAV:HSPA4_C12 = 0.04740 iii) AAV:HSPA4_C11 = 0.00341; AAV:HSPA4_C12 = 0.01095.