Generation of an endogenously HaloTagged TDP-43 iPSC line.

A) Schematic of knock in to N-terminus of TDP-43. Cas12a-crRNA ribonucleoparticle (RNP) and HaloTag donor plasmid containing homology arms to TDP-43 were nucleofected into iPSCs. Halo was knocked in to a single TDP-43 allele at the N-terminus of the protein, before exon 2.

B) Microscopy validation of Halo-TDP-43 localization to nucleus. Halo in pink, ConA lipid dye in green, and Hoechst in blue. Scale bar 10 µm.

C) TDP-43 western blot of NT and TDP-43 KD neurons. Halo-TDP-43 is approximately 33 kDa heavier than untagged TDP-43, which corresponds to the molecular weight of Halo. RPL24 is shown as a loading control. TDP-43 KD decreases levels of both tagged and untagged TDP-43.

D) Quantification of western blot shown in (C) shows untagged (left) and Halo-Tagged (right) TDP-43 is decreased upon TDP-43 KD. Interestingly, Halo-TDP-43 is expressed at levels about one half as much as untagged TDP-43.

CRISPRi screen identifies modifiers of TDP-43 protein levels in iPSC-derived neurons.

A) Schematic of FACS screen. Halo-TDP-43 iPSCs were transduced with the dual guide library and selected. After selection, iPSCs were differentiated to neurons via Dox-inducible NGN2 expression. Neurons were FACS sorted and populations expressing high Halo-TDP-43 and low Halo-TDP-43 were collected, DNA extracted, and sgRNA libraries sequenced.

B) Rank plot of screen results showing genes whose KD increases Halo-TDP-43 levels (left) and genes whose KD decreases Halo-TDP-43 levels, including TDP-43 itself (right). BORC genes are in blue.

C) GO analysis of screen hits that reduce Halo-TDP-43 levels include the BORC complex and many mitochondria-associated terms. The FDR is from the calculated permutation p value of 1000 iterations.

D) GO analysis of screen hits that increase Halo-TDP-43 levels include many hits related to mRNA processing. The FDR is from the calculated permutation p value of 1000 iterations.

Meta-analysis of CRISPRi screens identifies novel and common genes.

A) Comparison of CellRox/Halo-TDP-43 meta-analysis to Liperfluo/Halo-TDP-43 meta-analysis shows many genes lie close to a line with a slope of one, demonstrating that many genes change levels of various readouts in similar ways. R value = 0.56. BORC genes are highlighted in blue, novel hits from Liperfluo and CellRox screens as compared to our Halo-TDP-43 screen are indicated in green and orange, respectively.

B) GO analysis of genes that negatively modulate readouts for CellRox and Halo-TDP-43 screens show DNA and signaling related terms. The FDR is from the calculated permutation p value of 1000 iterations.

C) GO analysis of genes that positively modulate readouts for CellRox and Halo-TDP-43 screens show translation related terms. The FDR is from the calculated permutation p value of 1000 iterations.

D) GO analysis of genes that negatively modulate readouts for Liperfluo and Halo-TDP-43 screens show terms related to ubiquitination, neddylation, and lipids. The FDR is from the calculated permutation p value of 1000 iterations.

E) GO analysis of genes that positively modulate readouts for Liperfluo and Halo-TDP-43 screens show adenosine, acetylation, and autophagy related terms. The FDR is from the calculated permutation p value of 1000 iterations.

Validation of modulators of TDP-43 levels in iPSC-derived neurons.

A) Schematic of hit validation. As a first validation, Halo-TDP-43 iPSCs were transduced with single sgRNAs against hit genes and Halo levels were analyzed by microscopy, generating a list of Halo-validated hits. Then some genes were selected from the Halo-validated hits and untagged iPSCs were transduced with the same virus and TDP-43 protein levels assessed via TDP-43 immunofluorescence microscopy, identifying hits not affected by a HaloTag.

B) Representative images of Halo-TDP-43 live cell imaging with BORCS6 KD. sgRNA plasmids contain a cytoplasmic BFP, enabling identification of cells expressing the sgRNA. For BORC genes, iPSCs were transduced with a lysosome marker (LAMP1-mApple) to ensure functional BORC KD through depletion of neuritic lysosomes. Scale bar represents 20 µm.

C) Quantification of BORC KD Halo-TDP-43 microscopy. All BORC genes tested (S1-S8) showed statistically significant decreases in Halo-TDP-43 levels compared to a non-targeting (NT) guide, indicated by blue dots. N=12 wells per genotype, 9 images per well (small gray dots). Significant p-values indicated on graph.

D) (D-G) Summary graphs of screen, Halo live cell imaging, and TDP-43 immunofluorescence imaging fold changes for D) BORC KD,

E) E) Ubiquitin-associated gene KD,

F) F) m6A-associated gene KD, and

G) G) mitochondria gene KDs. Color indicates strength of log2FoldChange for each condition, circle size indicates -log10p-value; values below 1.3 are not significant, corresponding to a p-value of 0.05. No circle indicates the gene was not tested in that experiment (i.e., not all genes were tested by immunofluorescence).

Endogenous validation of TDP-43 level modifiers in iPSC-derived neurons.

A) Quantification of BORC KD TDP-43 immunofluorescence microscopy. Both BORC genes tested (S2 and S6) show significant decreases in untagged TDP-43 levels compared to a NT guide, indicated by blue dots. N=4 wells per genotype, 9 images per well (small gray dots). Significant p-values indicated on graph.

B) Representative images of TDP-43 immunofluorescence imaging with BORCS6 KD. sgRNA plasmids contain a cytoplasmic BFP, enabling identification of cells expressing the sgRNA. Scale bar represents 20 µm.

C) Schematic of pLentiCRISPR plasmid. pLentiCRISPR enables lentiviral delivery of a plasmid expressing an sgRNA of interest under a U6 promoter and Cas9 under an Ef1alpha promoter. This enables knockout of a gene of interest targeted by an sgRNA.

D) Representative images of BORCS7 KO TDP-43 IF with lysosomes stained with anti-H4A3 antibody. Scale bar is 20 µm.

E) Quantification of TDP-43 immunofluorescence on BORCS7 knockout neurons. Both BORCS7 guides significantly reduced the amount of TDP-43 IF signal compared to a NT guide, light blue dots. N=10 wells, 9 images per well (light gray dots). Significant p-values indicated on graph.

TDP-43 protein levels require active and neuritically localized lysosomes.

A) Drugs targeting different aspects of lysosome function decrease Halo-TDP-43 levels. All drugs tested were significant compared to a DMSO treatment, indicated by red dots. N=12 wells, 9 images per well (small grey dots), mean of each well is indicated by large dot.

B) Representative images of drug treatments that decrease Halo-TDP-43 levels. Scale bar is 20 µm.

C) Representative 30 s kymographs of Halo-TDP-43, lysosomes (LAMP1-mApple), mitochondria (mito-mEmerald), showing co-transport of Halo-TDP-43 with organelles. Green arrowhead indicates co-localization with static mitochondria; blue arrows indicate co-localization with motile lysosomes. Scale bar is 10 µm.

D) Quantification of Halo-TDP-43 signal with organelles. Motile Halo-TDP-43 in neurites is primarily co-transported with lysosomes or lysosomes and mitochondria together. Stationary Halo-TDP-43 is more likely to be associated with mitochondria.

E) Upon BORCS7 KD, TDP-43 is less mobile compared to a NT guide, likely due to restriction of lysosomes to the soma. N=4 wells (mean per well indicated by large dot), 8 images per well (small dots).

BORC KD does not affect TDP-43 RNA levels.

A) qPCR of NT, BORCS6 KD, and TDP-43 KD neurons showing decreased TDP-43 mRNA levels in TDP-43 KD, but not BORCS6 KD. Normalized to PPIA. NT n=3, BORCS6 KD n=4, and TDP-43 KD n=4.

B) Normalized transcript counts of NT and BORCS6 KD neurons showing about half as much BORCS6 mRNA in BORCS6 KD neurons. NT n=4, BORCS6 KD n=3.

C) Normalized transcript counts of NT and BORCS6 KD neurons showing no change in TARDBP (TDP-43) mRNA levels in BORCS6 KD neurons. NT n=4, BORCS6 KD n=3.

D) Volcano plot comparing BORCS6 KD with NT neurons. 98 genes increase expression upon BORCS6 KD (right of graph), while 139 genes decrease expression (left of graph). NT n=4, BORCS6 KD n=3.

E) GO BP enrichment analysis from Enrichr of genes significantly altered in BORCS6 KD neurons compared to NT neurons shows several neuron and axon related terms.

BORC KD alters the abundance and half-lives of TDP-43 and other proteins.

A) TDP-43 protein has decreased total abundance in day 7 BORCS6 neurons.

B) Volcano plot of total protein abundance comparing BORCS6 KD to a non-targeting guide. Some proteins (purple) have increased abundance, while a larger number (teal) have decreased abundance. Non-significant or low log2(fold change) proteins are indicated in grey. Vertical lines at log2(fold change) of 1 and -1, horizontal line at p-value = 0.05.

C) TDP-43 has longer half-life in day 7 BORCS6 KD neurons.

D) Volcano plot of half-life changes in BORCS6 KD neurons as compared to neurons expressing a non-targeting guide. Non-significant or low log2(fold change) protein turnovers are indicated in grey. Horizontal line at p=0.05, vertical lines at log2(fold change) of 0.5 or -0.5.

E) Density plot of global protein turnover shows longer protein half-lives upon BORC KD.

F) Gene ontology enrichment analysis using proteins with longer half-lives in BORCS6 KD neurons shows KEGG pathways related to protein export, proteasome, and metabolism.