The amount of TDP-43 expressed in neurons is tightly regulated and TDP-43 undergoes autoregulation of its own transcript levels¹⁴ to ensure levels are maintained at appropriate levels. To visualize and quantify the amount of TDP-43 in living cells, we used CRISPR technology to endogenously tag the N-terminus of TDP-43 with a HaloTag (Figure 1A). We selected a clone with correct editing based on sequencing. In this clone, knock in of the HaloTag was heterozygous, leaving the second allele of TDP-43 intact and unedited. The lead clone displayed a normal karyotype (Figure S1A), demonstrating that there were no major chromosomal abnormalities introduced during the gene editing or subsequent clonal expansion. Imaging of iNeurons differentiated from the knock in iPSC line showed the majority of Halo signal in the nucleus, as is expected for TDP-43 localization (Figure 1B). A western blot against TDP-43 shows a shift of approximately 35 kDa in the two TDP-43 positive bands, corresponding to the molecular weight of Halo (33 kDa) (Figure 1C, S1B). Knocking down TDP-43 using CRISPRi reduced the amount of both tagged and untagged TDP-43 compared to a non-targeting (NT) sgRNA (Figure 1C-D). Expression of Halo-TDP-43 was roughly 60% of endogenous TDP-43, indicating that the Halo-tagged protein is either not transcribed or translated as efficiently as untagged protein, or the protein is degraded more readily.

To identify upstream modifiers that alter TDP-43 levels, we performed a genome-wide CRISPRi FACS-based screen in both Halo-TDP-43 iPSCs and iNeurons differentiated from our Halo-TDP-43 iPSC line using a dual sgRNA library, enabling 1000x coverage of the genome by sorting only 20 million iNeurons¹². We sorted for cells highly expressing the GFP sgRNA marker and then sorted the cells containing the lowest 25% of Halo signal into one tube and the cells containing the highest 25% of Halo signal into another (Figure 2A). sgRNAs targeting the TARDBP transcription start site decreased Halo-TDP-43 levels in both the iPSC and iNeuron screens (Figure 2B and S2A, Tables S2 and S4), indicating that our screen successfully identified genes that decrease TDP-43 levels. Non-targeting guides are clustered in the middle of the rank plot for each screen, showing that gene knockdown is needed to alter Halo-TDP-43 levels (Figure 2B and S2A). Among genes whose knockdown decreases Halo-TDP-43 levels, we identified all eight components of the BORC complex, which regulates anterograde lysosome transport in axons as hits in the iNeuron, but not the iPSC screen (Figure 2B-C and S2A, Tables S2 and S4). Knockdown of genes related to mitochondrial function also decrease Halo-TDP-43 levels (Figure 2C and S2B). Further, we identified genes whose knockdown increases Halo-TDP-43 levels, including genes involved in RNA processing (Figure 2D and S2C, Tables S1 and S3). In comparing the top GO terms associated with genes that increase or decrease Halo-TDP-43 levels, we found only one hypergeometric test (HGT) term associated with both the neuron and iPSC screens, nonsense mediated decay (NMD) independent of the exon junction complex (EJC), although it is associated with increased Halo-TDP-43 levels in iNeurons and decreased Halo-TDP-43 levels in iPSCs (Figure S2D-G). Thus, our hits are cell-type specific.

CRISPRBrain (https://crisprbrain.org/) contains datasets from many CRISPRi and CRISPRa screens on different cell types examining various phenotypes. We identified three FACS-based CRISPRi screens on iNeurons that identify modifiers of reactive oxygen species (CellRox) and lipid peroxidation (Liperfluo) using fluorescent indicators¹¹ and tau aggregates¹⁵. We performed an unbiased fixed effects meta-analysis of these screens with our Halo-TDP-43 screen to determine if there were any common hits across screens. These common hits across multiple screens may be real hits that simultaneously change TDP-43 and alter ROS-levels, lipid peroxidation, or tau aggregation; however, they may also be “false-positives” that are altering general proteostasis, general cellular stresses, or neuron differentiation, where phenotypes examined in these screens are a secondary or indirect effect of knockdown. Interestingly, comparison of z-scores between screens shows many genes that either positively or negatively regulate both our screen and the CRISPRBrain screens (Figure 3A-F). With the increased power from the multiple screens, we were able to identify novel hits that were underpowered previously (Figure 3A-F). Genes were considered “hits” if they were novel with directionality (either up or down) compared to the total population (the average of the hits in the CRISPRBrain screens). For the Halo-TDP-43 screens, genes were filtered by a LFC of 1 or -1 compared to the total population. We performed GO analysis on common hits from the meta-analysis across screens and found enrichment in many interesting categories, including ubiquitination, neddylation, autophagy, protein targeting, cell division/differentiation, and cytoskeletal related terms (Figure S3A-F). On the other hand, some hits, like the BORC components, seem to be specific to our Halo-TDP-43 screen, so we chose to focus downstream analysis primarily on these hits that uniquely regulate TDP-43 biology. While this analysis provides a level of validation of our screen results, we picked genes from select categories to do a secondary, microscopy-based screen to further validate their effect on TDP-43 levels.

We cloned single sgRNA plasmids for 59 genes (Table S5) that either increased or decreased Halo-TDP-43 in iNeurons but not in iPSCs. sgRNAs were transduced into iPSCs individually, followed by differentiation of iPSCs into iNeurons. We used automated microscopy and analysis to visualize and quantify the Halo-TDP-43 signal in cells after each gene knockdown (Figure 4A). 34 of 59 (58%) tested genes altered Halo-TDP-43 levels in the microscopy validation (Figure 4B-G, S4A-D, Table S5). For every gene tested except one, we observed a simple increase or decrease in Halo-TDP-43 levels without an obvious change in Halo-TDP-43 localization (Figure 4B). Since we used a single sgRNA plasmid for validation and not the dual guide plasmid used in the screen, some of the genes that did not pass this stage of validation may not have had sufficient gene knockdown in the validation to elicit the effect observed in the screen. However, we were interested primarily in the genes whose knockdown had the strongest effect on Halo-TDP-43 levels. To determine if results from Halo-TDP-43 expression assays also applied to endogenous, untagged TDP-43 levels, we selected 22 genes from different groups in our list that passed Halo validation and performed immunofluorescence microscopy for endogenous (untagged) TDP-43 (Figure 4D-G, 5A-B, S4E-F, Table S5). Of these, 18 (82%) gene knockdowns showed changes in endogenous TDP-43 levels (Figure 4D-G, S4E-F, Table S5). However, many gene KDs, particularly genes involved in ubiquitination and m6A RNA methylation, changed untagged TDP-43 levels in the opposite direction as they change Halo-TDP-43 levels (Figure 4D-G, Table S5). We posit that this Halo-untagged difference may be due to inherent destabilization of the HaloTagged TDP-43. KD of BORC subunit genes changed TDP-43 levels in the same direction for both Halo imaging and immunofluorescence (Figure 5A-B). To rule out the possibility of neighboring gene or off-target effects of CRISPRi, as has been reported previously¹⁶, we examined the impact of BORC knockout (KO) on endogenousTDP-43 levels. Using the pLentiCRISPR system, which expresses the sgRNA of interest on the same plasmid as an active Cas9¹⁷, we found that KO of BORCS7 using two different sgRNAs decreased TDP-43 levels by immunofluorescence (Figure 5C-D). Because of the known relationship between lysosomal dysfunction and FTD/ALS pathophysiology^18-24, we focused on the relationship between TDP-43 levels, lysosomal biology, and BORC.

As lysosomes are the primary recycling center of the cell, breaking down macromolecules into their subunits, we first asked whether lysosome activity was required to maintain TDP-43 levels in iNeurons as has been observed in other cell types^{25, 26}. We treated Halo-TDP-43 neurons with various drugs that disrupt distinct processes in the lysosome pathway and asked if Halo-TDP-43 levels changed. Chloroquine (decreases lysosomal acidity), CTSBI (inhibits cathepsin B protease), ammonium chloride (NH₄Cl, inhibits lysosome-phagosome fusion), and GPN (ruptures lysosomal membranes) all consistently decreased Halo-TDP-43 levels, while Bafilomycin A1 (BafA1, prevents lysosomal acidification and fusion with autophagosomes), LLOME (causes lysosomal membrane rupture), and apilimod (PIKfyve inhibitor) did not cause changes in Halo-TDP-43 levels (Figure 6A-B, S5A-C). The varied mechanisms of action of these drugs indicated that many lysosomal perturbations could alter TDP-43 levels. Further, two protein translation inhibitors, anisomycin and cycloheximide (CHX) also decreased Halo-TDP-43 levels (Figure 6A, S5B).

Having shown that functional lysosomes are required to maintain Halo-TDP-43 levels, we next tested the role of lysosome location in iNeurons. As the BORC complex is required for anterograde movement of lysosomes within axons of neurons^{13, 27}, we first asked whether Halo-TDP-43 is transported on lysosomes. Our previous work showed that mRNA transport granules can be transported on lysosomes and that overexpressed TDP-43 co-trafficked with lysosome-associated transport granules²⁸. Indeed, in comparing motile Halo-TDP-43 with static Halo-TDP-43 in neurites, we found that motile TDP-43 was most often associated with lysosomes, not mitochondria (Figure 6C-D). Intriguingly, upon restricting lysosomes to the soma by knocking down a BORC subunit, we find that the fraction of motile Halo-TDP-43 in the periphery is reduced (Figure 6E, S5D).

We next asked whether TDP-43 RNA levels were also changed. Using qPCR, we found TDP-43 mRNA levels were unchanged or slightly increased upon BORCS6 KD (Figure 7A, S6A). As expected, we observed statistically significant reduction of TDP-43 mRNA upon TDP-43 KD via CRISPRi (Figure 7A, S6A). We then performed total RNA sequencing of BORCS6 KD iNeurons. While BORCS6 mRNA levels were reduced by ∼50% (Figure 7B), no change in TDP-43 mRNA levels occurred (Figure 7C), indicating that loss of BORC likely decreases TDP-43 protein levels independent of TDP-43 transcript expression.

As loss of BORC drastically changes the axonal transcriptome, particularly for mitochondrial genes²⁹, we wondered what the effect of BORC KD would be on the whole cell transcriptome. We found that NT and BORCS6 KD samples were substantially separated on a PCA plot, with over 90% of the variability between the samples being explained by a single principal component (Figure S6B). We found 98 upregulated genes and 139 downregulated genes (Figure 7D) in BORCS6 KD neurons. GO analysis revealed an enrichment of terms related to neuron development and axon outgrowth (Figure 7E). As loss of TDP-43 can cause splicing changes and the inclusion of cryptic exons^30-32, we asked whether BORCS6 KD decreased TDP-43 levels sufficiently to induce inclusion of cryptic exons^{33, 34}. We did not observe an increase in expression of cryptic exons in genes that are commonly mis-spliced in iPSC-derived neurons after TDP-43 loss, including STMN2 and UNC13A. As such, BORCS6 KD does not reduce TDP-43 protein levels sufficiently to cause functional loss of splicing repression. As TDP-43 alternative poly adenylation (polyA) contributes to TDP-43 autoregulation and levels¹⁴, we asked whether BORCS6 KD changed polyA site usage using REPAC³⁵. We found many sites changed upon BORCS6 KD (Figure S6C), with these genes relating to GO Biological Process terms like splicing, catabolism, and transport/localization (Figure S6D-E). However, we did not observe any changes in TDP-43 polyA site usage upon BORCS6 KD (Figure S6C), so alternative polyA cannot explain the changes in TDP-43 protein levels.

As loss of BORC prevents the transport of lysosomes to axons^{27, 29} and mRNAs can be transported on lysosomes to the periphery²⁸, we speculated that TDP-43 levels could be decreased in BORC KD iNeurons due to decreased axonal translation of TDP-43 mRNA. Using a TDP-43 mRNA N-terminally tagged with SunTag epitopes and with PP7 stem loops after the 3’ UTR, we used microscopy to quantify the co-localization of TDP-43 new protein synthesis (SunTag signal) with mRNA (PP7-coat protein signal)³⁶. Surprisingly, we did not observe a change in TDP-43 translation (Figure S7A). As such, we hypothesized that loss of BORC might alter TDP-43 protein degradation rather than its synthesis. To test this hypothesis, we performed dynamic stable isotope labeling by amino acids in cell culture (dSILAC) to measure protein half-lives as well as whole neuron proteomics to measure protein abundances in BORCS6 KD and non-targeting iNeurons. We found that TDP-43 total protein levels are decreased in BORCS6 KD neurons (Figure 8A), further validating our earlier microscopy findings. We observed a similar trend for many proteins in the proteome, with more proteins showing decreased abundance than increased (Figure 8B). Interestingly, TDP-43 shows longer half-life in BORCS6 KD neurons compared to the NT guide (Figure 8C), suggesting that protein degradation is potentially decreased in BORC KD iNeurons. We observed similar results for the total proteome, with many proteins showing longer turnover at day 7 in BORCS6 KD neurons compared to NT neurons (Figure 8D-E). Longer half-lives were also observed in d15 neurons although the changes in both abundance and half-life were reduced (Figure S7B-E), suggesting the neurons may be better able to compensate for the loss of BORC at later time points. In comparing the KEGG and REACTOME terms shown as enriched in our proteins with longer turnover times in BORCS6 KD neurons (Figure 8F, S7F) to the mRNAs whose localization is altered in BORC KO neurons²⁹, we observe many of the same categories are enriched, including ribosome and biosynthetic pathways. We further queried our data for posttranslational modifications (PTMs). We were able to identify 1333 phosphorylation sites. No PTMs were identified on TDP-43 at either time point, so PTM enrichment may be needed to query the role of PTMs in regulating the change in TDP-43 half-life with BORCS6 KD. We were able to detect 10 proteins with significantly changed phosphosites upon BORCS6 KD: KRI1 and RALY have phosphosites increased upon BORCS6 KD while OXSR1, ELAVL4, MAP4, ADD2, CSNK2B, FIP1L1, and RSF1 have phosphosites decreased upon BORCS6 KD at d7 (Figure S7G). At d15, only BASP1 showed a significantly decreased phosphosite (Figure S7H). interestingly, ADD2, CSNK2B, MAP4, OXSR1, and RALY are detected to have differential phosphorylation and increased half-life, suggesting these phosphosites may contribute to regulation of the turnover of these proteins. As TDP-43 and the proteome show similar effects of BORCS6 KD on protein half-life, iNeurons are likely in steady state and making as much new heavy protein as they are degrading the light protein. As such, protein translation or protein degradation could be reduced. Although we did not observe a change in TDP-43 translation in our SunTag experiment, it may not be accurately reflecting the translation levels of TDP-43 due to the experimental necessity of over-expressing tagged TDP-43. As such, BORC may be affecting TDP-43 protein levels through a complicated mechanism.

Procurement and use of WTC11 cells from the Coriell cell repository followed all policies of the NIH Intramural research program. WTC11 cells were derived from an apparently healthy 30-year-old male. iPSC culture was performed following previously described protocols⁴⁸. Briefly, iPSCs were grown in Essential 8 Medium (Thermo Fisher Scientific, A1517001) on tissue culture plates that had been coated with Matrigel (Corning, 354277). Medium was replaced daily or as needed. Accutase (Thermo Fisher Scientific, A1110501) was used to dissociate cells for passaging as single cells. 50 nM Chroman-1 (MedChemExpress, HY-15392) was added to promote survival after thaw and accutase treatment, until cell colonies had expanded to at least 4-5 cells/colony. The iPSCs used here were previously engineered to express mouse neurogenin-2 (mNGN2) with a doxycycline inducible promoter for neuronal differentiation (i11w-mN)⁴⁹. Many experiments used a line that additionally expressed a catalytically inactive dCas9 fused to a KRAB transcriptional repression domain for CRISPRi knockdowns¹⁰. These engineered cells containing dCas9 are referred to here as i11w-mNC iPSCs.

iNeuron differentiation and culture was performed following previously described protocols⁴⁸. Briefly, on day 0 of neuronal differentiation, iPSCs were passaged with accutase and plated on Matrigel-coated tissue culture plates. iPSCs were plated in induction media (knockout DMEM/F12 media (Life Technologies Corporation, #12660012) supplemented with N2 supplement (Life Technologies Corporation, 17502048), 1x GlutaMAX (Thermo Fisher Scientific, 35050061), 1x MEM nonessential amino acids (NEAA) (Thermo Fisher Scientific, 11140050), 50 nM Chroman-1, and 2 μg/mL doxycycline (Clontech, 631311)). Media was changed daily. On day 3 of neuronal differentiation, cells were passaged with accutase and plated on tissue culture plates that were coated with poly-L-ornithine (Sigma-Aldrich, P3655). Cells were plated in maturation media (BrainPhys media (Stemcell Technologies, 05790) supplemented with B27 Plus Supplement (Thermo Fisher Scientific, A3582801), 10 ng/mL BDNF (PeproTech, 450-02), 10 ng/mL NT-3 (PeproTech, 450-03), 1 μg/mL mouse laminin (Sigma, L2020-1MG), 50nM Chroman-1 (MedChem Express, HY-15392), and 2 μg/mL doxycycline (Clontech, 631311)). Half-media changes were performed every other day or every 3 days. Unless otherwise specified, all experiments were performed on day 7 neurons.

1 million iPSCs were transfected with premixed 20 µg Alt-R™ A.s. Cas12a (Cpf1) V3 (IDT # 1081068), 200 pmol crRNA 5’-GGAAAAGTAAAAGATGTCTGAAT (IDT), and 10 µg knockin (KI) plasmid donor (gene-synthesized and cloned into pUC57 by Genscript; synthesized sequence contains HaloTag coding sequence inserted immediately downstream TDP-43 start codon ATG and flanked by 400 bp left and right homology arms) using Nucleofector 4D with buffer P3 and program CA-137 (Lonza) and then plated onto one well of rhLamin521 (Thermo) coated 6-well plate with StemFlex, RevitaCell (Thermo) and HDR enhancer V2 (IDT). Transfected cells were cultured in 32°C incubator for 3 days before moving to 37°C incubator, and fresh medium was changed daily after transfection. One week after transfection, the iPSCs were live stained with Oregon Green (Promega #G2801) following the manufacturer’s protocol. Positive cells were sorted into 96-well Matrigel-coated plate at 1 cell/well with 100 µl/well StemFlex and 1x CloneR2 (Stem Cell Technologies). 10-12 days later, single-cell clones were picked and KI clones were confirmed by 5’-KI and 3’-KI junction genomic PCR (5KI-F: 5’-ATCGACTGGGACCTATCACG, 5KI-R: 5’-GCGTACCCTCGATAAAAACG; 3KI-F: 5’-GAGACCTTCCAGGCCTTCC, 3KI-R: 5’-TCTACAATCCCCAGTTTCCA). Heterozygous KI clones were further identified by the presence both non-KI and KI allele using primers recognizing homology arms (WT-F: 5’-GACGCATCATAAGCCTTCAG, WT-R: 5’-CCATGGATGAGACACACACC). Both non-KI and KI alleles were subject to Sanger sequencing to exclude heterozygous KI clones with indels in non-KI alleles.

Approximately 50 million Halo-TDP-43 iPSCs were transduced with a whole genome dual guide library¹² to a titer of approximately 70% in E8 with Chroman 1. The next day, the media was changed to E8 without chroman 1. The next day, iPSCs were split onto new Matrigel coated plates in E8 with chroman 1. The day following the split, iPSCs were treated with 8 µg/mL puromycin in E8 Chroman 1 to select for cells expressing an sgRNA. After selection, cells were split and differentiated into neurons following the standard protocol above. Some cells were maintained as iPSCs for the iPSC screen. On day 3 post dox induction, neurons were split onto final PLO-coated plates. On day 7 post dox induction, cells were split for FACS. Cells were stained with 200 µg/mL Halo Ligand 646 (Promega HT1060) for 15 minutes in appropriate media. Papain solution (Worthington LK003176) was prepared by adding 20 mL TrypLE without phenol red (Gibco 12604-013) to one vial of papain and placing it at 37°C for 10 minutes. Neurons were washed with PBS and then washed twice with PBS with 0.5 mM EDTA. Papain/TrypLE was added to cells for 1-2 minutes at 37°C. Papain was removed without disturbing the neuron sheet and trituration solution (Neuron culture media, 10 µM ROCK inhibitor, and one vial of DNAse freshly dissolved) was added to cells before transferring them to a conical tube. Cells were pipetted 3-10 times in the conical tube until cell clumps were gone. PBS was added to cells and cells were pelleted by centrifugation at 200 xg for 5 minutes. Neurons were resuspended in 1 mL Papain Inhibitor Media (27 mL trituration media with 3 mL Papain inhibitor) and incubated for 5-10 minutes. Cells were again pelleted by centrifugation at 200 xg for 5 minutes and resuspended in cell culture media before transferring cell suspension into a 5 mL collection 12x75 mm polypropylene FACS tube with 35-40 µm strainer cap. Cells were kept on ice until FACS was performed. Cells were sorted using the MoFlo Astrios cell sorter (Beckman Coulter), with Summit software (Beckman Coulter) to setup a sorting template. GFP positive cells were detected using the 488 nm laser excitation, and 520/30 nm filter for capturing signal emission. Halo was detected using 640 nm laser excitation and 675/30 nm filter for emission. The cells were first gated on GFP expression, which served as a highly expressing co-expression marker for sgRNA, and then gated on Halo expression, as follows. Four groups were collected from the FACS session: the bottom 25% of iPSCs expressing Halo-TDP-43, the top 25% of iPSCs expressing Halo-TDP-43, the bottom 25% of neurons expressing Halo-TDP-43, and the top 25% of neurons expressing Halo-TDP-43. Genomic DNA was collected from each fraction using the NucleoSpin Blood Kit (Macherey Nagel 740950.50) following manufacturer’s instructions. Briefly, cells were washed in PBS and resuspended in PBS containing proteinase K. Next BQ1 was added to each tube and mixed by vortexing. Samples were incubated at 56°C for 15 minutes and cooled to room temperature for at least 30 minutes. 100% ethanol was added to the tube and mixed by vortexing before loading onto the column. Columns were spun for 3 minutes at 4000 xg and flow through discarded. BQ2 was added to the column and columns spun for 2 minutes at 4000 xg. BQ2 step was repeated a total of 2 times. To elute genomic DNA, pre-heated BE was added to each column, incubated for 5 minutes at room temperature, and spun for 4 minutes at 4000 xg in a new tube. Elution was repeated a second time. Next, sgRNA amplicons were enriched using primers containing indexing barcodes and Illumina adapters. 10 µg of genomic DNA was used per PCR reaction. PCR reactions also contained 100 µM of one of the following indexed forward primer (caagcagaagacggcatacgagatcgctcagttctgctatgctgtttccagcttagctcttaaac, caagcagaagacggcatacgagattatctgaccttgctatgctgtttccagcttagctcttaaac, caagcagaagacggcatacgagatatatgagacgtgctatgctgtttccagcttagctcttaaac, caagcagaagacggcatacgagatcttatggaattgctatgctgtttccagcttagctcttaaac), 100 µM of one of the following indexed reverse primer (aatgatacggcgaccaccgagatctacactcgtggagcgagcacaaaaggaaactcaccctaactgt, aatgatacggcgaccaccgagatctacacctacaagataagcacaaaaggaaactcaccctaactgt, aatgatacggcgaccaccgagatctacactatagtagctagcacaaaaggaaactcaccctaactgt, aatgatacggcgaccaccgagatctacactgcctggtggagcacaaaaggaaactcaccctaactgt) and 1x NEBNext Ultra Q5 MasterMix (NEB M0544L). Cycling parameters were as follows: 98°C for 30 seconds, 22-24 cycles of 98°C for 10 seconds, 66°C for 75 seconds, then a 5-minute hold at 72°C after cycles and a hold at 4°C. PCR reactions were pooled and leaned up with a PCR purification. A 0.5X-0.65X SPRI cleanup as performed prior to sequencing. Half volume of SPRI beads (Beckman Coulter B23318) were added to the pooled PCR reaction and incubated at room temperature for 10 minutes. Beads were removed with a magnet and supernatant collected. Next, a 0.65 volume of SPRI beads were added to cleared supernatant, incubated at room temperature for 10 minutes and tubes placed on a magnetic stand. Supernatant was removed and discarded. Beads were washed with fresh 80% ethanol twice and beads air dried for 2-5 minutes. Sample was eluted in 30 µL EB buffer. The SRPI PCR cleanup was repeated in its entirety to remove excess primer contamination. PCR product yield, size, and quality was checked on a Bioanalyzer (Agilent) with a High Sensitivity DNA chip (Agilent B23318). Enriched PCR product is 562 base pairs in length. Samples were sequenced on the MiSeq with 1% PhiX through paired end 50 sequencing with custom primers (Read1: gtgtgttttgagactataagtatcccttggagaaccaccttgttgg, Read2: tgctatgctgtttccagcttagctcttaaac, i7 Indexing primer: aagagctaagctggaaacagcatagca). Sequencing files were demultiplexed based on the sample PCR. To remove recombined dual guides, fastq files were filtered for reads containing paired guides for the same target gene using the parser.py file (https://github.com/NIH-CARD). The parsed dual hits file was analyzed using the standard MAGeCKFlute robust ranked algorithm (RRA) pipeline⁵⁰ comparing the high 25% to low 25% of the TDP-43 HaloTag fluorescence (results in Tables S1-4). The RRA uses a negative binomial model to generate p-values to rank guides from negative to positive. Guide output files were filtered to remove low count guides and used to generate rank plots of target guides and non-targeting negative controls. All data and code for plots is available on (https://github.com/jhawrot/TDP_BORC/tree/main).

CRISPRi FACS screens in i³Neurons¹¹ were obtained from CRISPRBrain (https://crisprbrain.org/) and the Kampmann lab and underwent data harmonization with the screens presented here. First, screen phenotype scores and log fold changes were aligned by converting effect estimates to standardized beta values represented by z-scores. Next, extreme p-values were stabilized by truncation to prevent distortions in the statistical tests due to outliers or very small probabilities, ensuring a robust meta-analysis, accommodating the floating point of the system and acknowledging that this truncation will cause results to be more conservative. Standard errors of the effect estimates were derived by dividing the beta estimate by the absolute value of the z-scores. Fixed effect meta-analyses were then carried out for all possible screen combinations (combining already published versus novel screens). These functions calculate pooled estimates of effect sizes, variance, and test for heterogeneity among screens. Novelty was assessed by filtering associations that were not significant after multiple test corrections in either of the constituent datasets but were significant after false-discovery rate adjustment of the meta-analysis. Data was visualized using modified MAGeCKFlute pipelines to assess putative biological functions of significant genes using published enrichment functions including clusterProfiler, GOstats, enrich.HGT, and GSEA packages. TDP-43 expression hits were grouped into positive and negative modifiers using Log2FC values, and statistical significance was assessed by gene ratio or FDR depending on the enrichment analysis. Code is available on (https://github.com/NIH-CARD/V_Ryan_TDP43_meta_2024).

Individual CRISPRi knockdowns were performed by cloning sgRNAs into pU6-sgRNA EF1Alpha-puro-T2A-BFP (Addgene plasmid # 60955). sgRNA sequences for screen validation are listed in Table S5. To make lentivirus, Lenti-X human embryonic kidney (HEK) cells were split with trypsin, plated on poly-L-ornithine coated plates, and allowed to recover. Cells were then transfected with sgRNA plasmids on a 6 well plate using 3.75 µL Lipofectamine 3000 Reagent and 5 µL P3000 Enhancer Reagent (Invitrogen, L3000075), Opti-MEM (Gibco, 31985062), psPAX2 F46, pMD2G F45, and pAdVantage F44. Transfected cells were cultured for two days in DMEM supplemented with GlutaMAX--I (Thermo Fisher Scientific, 35050061) and 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, F4135). Media was then changed and supplemented with (1:500) viral boost reagent (ALSTEM, VB100), and cells were cultured for two days before media was harvested and Lenti-X Concentrator (Takara Bio USA, 631231) was added at (1:3) to concentrate the virus for 2-3 days at 4°C. Virus was then collected, aliquoted, and stored at -80°C until use.

iPSCs were transduced at scales of 0.1-1e6 cells per 100 µL concentrated virus while cells were in suspension. Cells were allowed to recover for two days before 10 µg/mL puromycin dihydrochloride (Sigma-Aldrich, P9620) was added for selection. For cells that were differentiated, transduction was performed four days before initiating neuronal differentiation.

Hits from the screen were validated by microscopy with individual sgRNA knockdowns in i11w-hT neurons. Cells were plated, transduced, and differentiated as described above. Fluorescent HaloTag ligand JF646 (Promega, GA112A) was added at 200 µg/mL approximately 15 minutes before microscopy. Cells were imaged on Nikon Ti2 with automated stage at 20x with exposure times of 100ms for all channels. Laser power was as follows: 640 nm at 100%, 405 nm at 25%, and 561 nm at 100%. Halo-TDP-43 fluorescent intensity was quantified using CellProfiler⁵¹ or Nikon Elements General Analysis 3 and graphed in GraphPad Prism.

sgRNAs for genes selected for further validation on untagged TDP-43 were transduced into i11w-mNC iPSCs and neurons differentiated as above. On day 7, neurons were fixed with 4% PFA (Thermo Fisher Scientific, 28906) for 10 minutes at room temperature. PFA was removed and cells washed in PBS. Cells were then permeabilized with 0.1% Triton-X 100 (Sigma-Aldrich, 93443) for 10 minutes before blocking in 2% bovine serum albumin (BSA) (Jackson ImmunoResearch, 001-000-162) for 10 minutes. TDP-43 primary antibody (Proteintech, 10782-2-AP) was diluted 1:1000 in 2% BSA and added to cells at 4°C overnight. After washing, Donkey Anti-Rabbit IgG (H+L), Highly Cross-Adsorbed secondary antibody (CF488, Biotium, 20015) was added at 1:1000 in 2% BSA for 1 hour at room temperature. Cells were washed and DRAQ5 nuclear stain (Thermo Fisher Scientific, 62251) was added at 1:1000 dilution in PBS for 5 minutes. DRAQ5 was washed out with PBS three times before imaging. Stained cells were imaged on Nikon Ti2 with automated stage at 20x and an exposure time of 100 ms for 640nm and 488nm and 200 ms for 405nm. Laser power was 100% for all channels. TDP-43 fluoresce intensity was quantified using Nikon Elements and graphed in GraphPad Prism.

i11w-hT iPSCs were cultured and differentiated into neurons. i11w-mNC, treated for 24 hours with DMSO, were used as a Halo-negative control. Microscopy was performed on day 7 of neuronal differentiation. Wells were individually treated with drugs by removing half the media and adding back an equal volume of media containing 2x final concentration of each drug, for timepoints as stated below. A volume of DMSO equal to the largest volume of drug was added at the earliest timepoint to wells as a negative control for treatment. Treatments were as follows: 50 µM Chloroquine (Selleck Chemicals, S6999) for 24 hours; 50 µM CA-074me (Selleck Chemicals, S7420), a Cathepsin B Inhibitor (CTSBI), for 6 hours; 30 mM Ammonium Chloride (NH₄Cl) (Sigma-Aldrich, A9434) for 5 hours; 500 nM Bafilomycin A1 (BafA1) (LKT Labs, B0025) for 4 hours; 1x Lysosome Protease Inhibitor Cocktail (20 µM E64 (Selleck Chemicals, S7379), 10 µM pepstatin A (Selleck Chemicals, S7381) and 50 µM leupeptin (Sigma-Aldrich, L2884); referred to in Figure S5B-C as Protease Inhibitor) for 4 hours; 1 mM L-Leucyl-L-Leucine methyl ester (hydrochloride) (LLOME) (Cayman Chemical Company, 16008) for 4 hours; 400 µM Gly-Phe-β-naphthylamide (GPN) (Cayman Chemical Company, 14634) for 1 hour; 500 µM Sodium (meta) arsenite (NaAsO2) (Sigma-Aldrich, S7400), 1 hours; 40 µM Anisomycin (Sigma-Aldrich, A5862) for 1 hour; and 0.1 mg/mL Cycloheximide solution (CHX) (Sigma-Aldrich, C4859) for 45 minutes. Fluorescent HaloTag ligand was added as described above 15 minutes prior to imaging. Cells were imaged on Nikon Ti2 with automated stage at 20x in 640nm with an exposure time of 100ms and 100% laser power. Halo-TDP-43 fluorescent intensity was quantified using Nikon Elements and graphed in GraphPad Prism.

i11w-hT iPSCs were transduced with fluorescent tags for mitochondrial targeted mEmerald and LAMP1 tagged with mApple. The cells were then transduced with either BORCS6 or BORCS7 knockdown and non-targeting control guides. BORCS6 knockdown cells were cultured and differentiated as described above. BORCS7 knockdown cells were differentiated as neurospheres. Fluorescent HaloTag ligand was added to day 7 BORCS6 knockdown neurons and day 10 BORCS7 knockdown neurospheres, as described above. Cells were imaged on Nikon Ti2 with automated stage at 60x and exposure times of 100 ms for all channels. Laser power was 100% for 640nm and 561nm. 405nm and 488nm lasers were set at 100% and 99.4%, respectively, for BORCS6 knockdown cells, and at 50% for BORCS7 knockdown neurospheres. DAPI image was taken once at the beginning of the video; GFP, Cy5, and RFP channels were captured over 30 seconds with no delay between frames and an approximate frames per second (FPS) of 1.16. Puncta were counted from kymographs of axonal segments made in ImageJ or Nikon Elements and graphed in GraphPad Prism.

qPCR was performed using TaqMan Real-Time PCR assays. RNA was extracted from BORC and TDP-43 knockdown and non-targeting control i11-mNC-derived neurons using the RNeasy Plus Mini kit (QIAGEN, 74134). RNA concentrations were measured with a DeNovix DS-11 FX+ 1uL Spectrophotometer, and 855 ng of RNA was used as input for reverse transcription. Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814). The qPCR reaction was performed with 2x TaqMan Universal PCR Master Mix (Thermo Fisher Scientific, 4304437) on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System. Probes were run singleplex. The Taqman probe for the target was TARDBP, (FAM-MGB Hs00606522_m1, Thermo Fisher Scientific, 4453320). Two endogenous controls were used: PPIA (VIC-MGB-PL Hs04194521_s1, Thermo Fisher Scientific, 4448484); and PGK1, TaqMan ID: Hs00943178_g1, VIC-MGB-PL, (Thermo Fisher Scientific, 4448484). Relative change in gene expression was examined using the 2^-(ΔΔCt) analysis method⁵². Significance was evaluated using an ordinary one-way ANOVA with the Šidák correction for multiple comparisons.

A non-targeting (AGAGTTACGTCGCTTCGATC) and two sgRNA targeting sequences against BORCS7 (1: CGCGATTACGTCAGTACCAC, 2: GATTACGTCAGTACCACAGG) were cloned into the lenti-CRISPR v2 plasmid (Addgene 52961) following the Zhang Lab protocol (https://media.addgene.org/data/plasmids/52/52961/52961-attachment_B3xTwla0bkYD.pdf). Briefly, the plasmid was digested with BsmBI-v2 (NEB R0739), the cut vector was dephosphorylated with NEB Quick CIP (NEB M0525), and digested and dephosphorylated vector was extracted from an agarose gel. Primers containing the sgRNA targeting sequences (Eurofins) were phosphorylated with T4 PNK (NEB M0201) and annealed before being ligated into the cut vector using 2x Ligation Master Mix (Takara 6023). After sequencing to confirm correct cloning, plasmids were transfected into Lenti-X HEK cells using standard protocols (see above). Virus was then transduced into i11w-mN iPSCs and selected with puromycin using standard protocols (see above). Selected iPSCs were differentiated using standard protocols (see above). On day 7 post dox induction, neurons were fixed and immunofluorescence performed as above.

TDP-43 coding sequence and 3’UTR were cloned into a plasmid expressing 24 SunTag repeats under an Ef1alpha promoter followed by 24 PP7 stem loops³⁶ (modified from Addgene 74928). This plasmid was transfected into iPSCs stably expressing the SunTag scFv tagged with HaloTag under an Ef1alpha promoter (modified from Addgene 60907), PP7 coat protein tagged to 3 SnapTags (synthesized), and mEmerald-tagged lysosomes (to validate BORC KD). iPSCs were transduced with sgRNA and differentiated as spheres before plating on an 8 well glass bottom µ-Slide (Ibidi, 80827). Neurospheres were imaged at d10 post dox induction on a Nikon Ti2 spinning disk confocal microscope at 60x with only neurites in the field of view. Exposure times were as follows: 300 ms for 640nm, 200 ms for 561, 200 ms for 488, and 100ms for 405. Laser power was at 50% for DAPI and 100% for all other channels. Images were quantified for co-localized PP7 and SunTag (SnapTag and HaloTag, respectively) puncta using Nikon Elements. Resulting data were graphed in GraphPad Prism.

RNA was extracted from day 7 BORCS6 knockdown and non-targeting control i11-mNC neurons using the Direct-zol-96 MagBead RNA kit (Zymo Research, R2103). Manufacturer protocol was followed, including the optional DNAase step, with volumes increased for an automated run on the KingFisher. Quantified with Qbit and frozen at -80°C. RNA quality was checked on a BioAnalyzer and Strand-Specific RNA-seq library preparation was performed with rRNA depletion using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760) and the NEBNext® Globin & rRNA Depletion Kit. (New England Biolabs, E7750). Samples were run on an Illumina NovaSeq X 10B on a 2 x 150 bp flow cell.

Gene length normalized counts per gene were generated and then within sample normalization using counts per million (cpm) was performed. Next, pedestalling using a value of 2 and log2 transformation were performed. Cyclicloess was used to normalize expression across samples followed by exploratory analysis to insepct for and remove outlier samples if present. If present, cyclicloess nomrlaization is repeated. Data post cyclicloess normalization is then noise modeled (CV∼mean) by experiment condition (NT v KD) and a noise threshold for the data defined. This noise threshold is then used to filter remove noise-biased genes with surviving genes then subject to differential testing using the appropriate test. After differential testing, all statistics are captured and exported along with confirmatory visual generation post differential gene selection. Pathway analysis was performed to identify enriched pathways and functions for each comparison performed as part of differential testing. Alternative poly adenylation was assessed using REPAC³⁵. Results that had an absolute compositional Fold Change (cFC) >= 0.25 and an FDR corrected p-value < 0.05 were considered Poly Adenylation Sites that had a significant shortening (negative cFC) or lengthening event (positive cFC) and were plotted using VolcaNoseR⁵³. GO analysis of alternative poly-adenylation was performed using ShinyGO⁵⁴.

For cryptic splicing analyses, sequencing files were aligned to GRCh38 reference genome (STAR v2.7.3a)⁵⁵. We ran MAJIQ (v2.1)⁵⁶ on aligned BAM files using a custom Snakemake (v 5.5.4)⁵⁷ pipeline (https://github.com/frattalab/splicing). A threshold of 10% difference in percent spliced in (PSI, ΔΨ) was used to identify a significant change between groups. We defined cryptic splicing as junctions with ΔΨ > 10% that were present in <5% of control samples or ΔΨ < -10% that were present in >10% of control samples. An additional pipeline developed previously³⁴ was used to visualize and categorize each mis-spliced junction as cryptic exon, exon skipping, intron retention, or canonical junction (https://github.com/NIH-CARD/proteogenomic-pipeline).

BORC knockdown and non-targeting control i11w-mNC cells were cultured and differentiated as described above until day 5 of neuronal differentiation. On day 5, half of the cultures were changed to heavy-SILAC media (“dSILAC samples”), which was DMEM for SILAC (Thermo Fisher Scientific, 88364) supplemented with B27 Plus Supplement (Thermo Fisher Scientific, A3582801), 10 ng/mL BDNF (PeproTech, 450-02), 10 ng/mL NT-3 (PeproTech, 450-03), 1 μg/mL mouse laminin (Sigma, L2020-1MG), 50 nM Chroman-1 (MedChemExpress Catalog #HY-15392), 2 μg/mL doxycycline (Clontech, 631311), 0.5 mM lysine, and 0.3 mM arginine. Heavy SILAC media was supplemented with L-Lysine:2HCl (13C6, 99%; 15N2, 99%) (Cambridge Isotope Laboratories, Inc., CNLM-291-H) and L-Arginine:HCl (13C6, 99%; 15N4, 99%) (Cambridge Isotope Laboratories, Inc., CNLM-539-H). Remaining cultures were maintained in light SILAC media made with L-Lysine monohydrochloride (SIGMA, L8662 and L7039) and L-Arginine monohydrochloride (SIGMA, A4599) to produce non-SILAC samples to evaluate changes in total protein abundance. Half-media changes were performed every other day or every 3 days. Cells were harvested on day 7 and day 15 post-dox induction.

Protein was extracted from day 7 and day 15 neurons for mass spectrometry as done previously⁵⁸. Neurons were gently washed with PBS, then SP3 lysis buffer (50 mM Tris-HCI (Thermo Fisher Scientific, 15568025), 50 mM NaCl (Thermo Fisher Scientific, 24740011), 1% SDS (Thermo Fisher Scientific, 15553027), 1% Triton X-100 (MilliporeSigma, X100), 1% NP-40 (Thermo Fisher Scientific, 85124), 1% Tween 20 (MilliporeSigma, P9416), 1% glycerol (MP Biomedicals, 800687), 1% Sodium deoxycholate (wt/vol) (MilliporeSigma D6750), 5 mM EDTA, 5mM Dithiothreitol (DTT) (Thermo Fisher Scientific, R0862), 5 KU Benzonase (Sigma-Aldrich, E8263), and 1× cOmplete ULTRA Protease Inhibitor Tablets (one tablet per 10mL of lysis buffer) (Sigma-Aldrich, 5892791001)) was added directly to the wells. Neurons were scraped into the buffer to aid with lysis. Proteins were alkylated with 10 mM Iodoacetamide (IAA) (Thermo FIsher Scientific, A39271) at room temperature in the dark, then reduced by adding 2 µL of 500 mM DTT to the lysate and incubating at 25°C for 30 min in the light. A Thermo Fisher KingFisher Apex was used to perform the automated SP3 protein enrichment protocol. This protocol uses SP3 beads: GE SpeedBeads (GE45152105050250) and of GE SpeedBeads (GE65152105050250) combined in a 1:1 ratio. The SP3 beads are washed in LC-MS grade water (Pierce, 51140) and reconstituted in 50% EtOH at a concentration of 4 mg/mL. Samples are diluted 1:1 with 100% EtOH, washed in 80% EtOH, and eluted in 50 mM ammonium bicarbonate with 20 µg/mL lyophilized trypsin/LysC (Promega, V5073). After completion of the KingFisher SP3 protocol, samples were shaken overnight at 37°C. The KingFisher was used to perform a bead removal protocol twice. Solvents were removed in a speed vac. Samples were stored dry at -20°C until resuspension in 2% acetonitrile (Thermo Fisher Scientific, 51101), 0.4% Trifluoroacetic acid (TFA) (Thermo Fisher Scientific, 85183), and 0.1% formic acid (Thermo Fisher Scientific, 28905).

Peptide concentration from each sample was measured on a DeNovix DS-11 FX+ 1uL Spectrophotometer and normalized to 0.5 µg/µL. LC-MS/MS experiments were performed on a Thermo Scientific Orbitrap Lumos Tribrid Mass Spectrometer couple with a Thermo Scientific Ultimate 3000 HPLC system. The injection volume for each sample was 2 µl. Peptides were separated on a Thermo Scientific ES902 column (particle Size: 2 µm, diameter: 75 µm, length: 25 cm). Mobile phase A contains 0.1% formic acid in LC-MS grade water, and Mobile phase B contains 0.1% formic acid in LC-MS grade acetonitrile. Mobile phase B was increased from 3% to 22% in 64 min, then from 22% to 36% in 8 min. LC-MS/MS data were acquired in data-dependent mode. The MS1 scans were performed in orbitrap with a resolution of 120K, a mass range of 375-1500 m/z, and an AGC target of 4 x 105. The quadrupole isolation window is 1.6 m/z. The precursor ion intensity threshold to trigger the MS/MS scan was set at 1 x 104. MS2 scans were conducted in the ion trap. Peptides were fragmented with the HCD method and the collision energy was set at 30%. The maximum ion injection time was 35 ms. MS1 scan was performed every 3 sec. As many MS2 scans were acquired within the 3-sec cycle.

Proteomic raw data were analyzed using MaxQuant software (v. 1.6.14)⁵⁹. A reviewed Swiss-Prot Homo sapiens database and a custom neuron-specific contaminant FASTA library⁶⁰ (available to download at https://github.com/HaoGroup-ProtContLib) were used for protein and peptide identifications with a 1% false discovery rate cutoff. A maximum of two missed cleavages were allowed. A maximum of three variable modifications were allowed for cysteine N-ethylmaleimide, methionine oxidation, and protein N-terminus acetylation. Precursor and fragment tolerances were set to 25 ppm. Contaminatn proteins and peptides were removed. Protein intensities from protein.txt output files were used for nonSILAC data analysis. dSILAC data was analyzed using a previously established workflow⁶¹. A standard multiplicity with two labels was enabled for SILAC data, and Lys8/Arg10 was selected as the heavy channel. For SILAC proteomics, only y-type product ions with up to two charges were used for quantification. The “peptide.txt” report was exported for subsequent analysis in R. Peptides from contaminant proteins and peptides with intensities below 1000 were removed. Peptides with heavy/light ratios lower than 0.01 or higher than 100 were also removed to avoid outlier half-life measurements. A single timepoint calculation was used to calculate peptide half-lives assuming a steady state of turnover: , where R is the heavy-to-light ratio and ts is the labeling time point after media switch as described previously⁶¹. Protein half-lives were then determined as the harmonic mean of half-lives from unique peptides belonging to each protein.

Phosphopeptides were identified in cells cultured exclusively in light media. We employed the FragPipe LFQ-phospho workflow to detect and quantify modified peptides from our spectral files. This workflow integrates MSFragger for peptide identification with match-between-runs enabled⁶², PTMProphet for precise mass modification localization⁶³, and Philosopher for false discovery rate filtering and reporting⁶⁴. Differential expression of phosphopeptides was subsequently assessed in R (version 4.4.3) using the limma package (version 3.62.2). A threshold of a 1 log-fold change combined with a Benjamini-Hochberg adjusted p-value of 0.05 was applied to define significantly differentially expressed phosphosites.