Genome-wide CRISPR screen identifies protein pathways modulating tau protein levels in neurons.

Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.

A Genome-wide CRISPR Screen Identifies ZCCHC14 as a Host Factor Required for Hepatitis B Surface Antigen Production.

A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes.

Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will. Our inducible mutant hPSC library can be used for multiple genome-wide CRISPR screens in a variety of hPSC-induced cell types. As proof of concept, we performed three screens for regulators of properties fundamental to hPSCs: their ability to self-renew and/or survive (fitness), their inability to survive as single-cell clones, and their capacity to differentiate. We identified the majority of known genes and pathways involved in these processes, as well as a plethora of genes with unidentified roles. This resource will increase the understanding of human development and genetics. This approach will be a powerful tool to identify disease-modifying genes and pathways.

Bile acid analogues are activators of pyrin inflammasome.

Bile acids are critical metabolites in the gastrointestinal tract and contribute to maintaining intestinal immune homeostasis through cross-talk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate that the more potent analogue 11-oxo-12S-hydroxylithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL-18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide CRISPR screen with compound induced pyroptosis in THP-1 cells, we identified that inflammasome activation by BAA473 is pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the pyrin inflammasome. We surmise that pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help to further unveil the importance of pyrin in gut homeostasis and autoimmune diseases.

CRISPR/Cas9-Based Chemogenomic Profiling in Mammalian Cells.

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. It is based on identifying cellular hypersensitivity and resistance caused by individual gene modulations with genome-wide coverage. Due to the requirement of bar-coded, genome-wide deletion collections, high-resolution experiments of this nature have historically been limited to fungal systems. Pooled RNAi reagents have enabled similar attempts in mammalian cells but efforts have been hampered by significant off-target effects and experimental noise. The CRISPR/Cas9 system for the first time enables precise DNA editing at defined loci in a genome-wide fashion. Here we present the detailed protocol that leverages the CRISPR/Cas9 system for chemogenomic profiling and target identification of diverse chemical probes.

TMEM41B is a novel regulator of autophagy and lipid mobilization.

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and ß-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization.

p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells.

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1-3. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells3-13. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction3. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations14, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

TRRAP is a central regulator of human multiciliated cell formation.

The multiciliated cell (MCC) is an evolutionarily conserved cell type, which in vertebrates functions to promote directional fluid flow across epithelial tissues. In the conducting airway, MCCs are generated by basal stem/progenitor cells and act in concert with secretory cells to perform mucociliary clearance to expel pathogens from the lung. Studies in multiple systems, including Xenopus laevis epidermis, murine trachea, and zebrafish kidney, have uncovered a transcriptional network that regulates multiple steps of multiciliogenesis, ultimately leading to an MCC with hundreds of motile cilia extended from their apical surface, which beat in a coordinated fashion. Here, we used a pool-based short hairpin RNA screening approach and identified TRRAP, an essential component of multiple histone acetyltransferase complexes, as a central regulator of MCC formation. Using a combination of immunofluorescence, signaling pathway modulation, and genomic approaches, we show that (a) TRRAP acts downstream of the Notch2-mediated basal progenitor cell fate decision and upstream of Multicilin to control MCC differentiation; and (b) TRRAP binds to the promoters and regulates the expression of a network of genes involved in MCC differentiation and function, including several genes associated with human ciliopathies.

Genome-wide CRISPR screen for PARKIN regulators reveals transcriptional repression as a determinant of mitophagy.

PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.

Transcranial Motor Evoked Potential Alarm Criteria to Predict Foot Drop Injury During Lumbosacral Surgery.

STUDY DESIGN: A retrospective cohort analysis. OBJECTIVE: This study aims to investigate whether waveform alterations in transcranial motor evoked potentials (TCMEPs) can reliably predict postoperative foot drop. SUMMARY OF BACKGROUND DATA: Nerve injury leading to foot drop is a potential complication of lumbosacral surgery. Very limited data exist on the use of intraoperative TCMEPs to identify iatrogenic foot drop. METHODS: We retrospectively reviewed neuromonitoring data from 130 consecutive spine surgeries with instrumentation involving L4-S1. TCMEP waveform analysis included amplitude (A), area under the curve (AUC), latency (L), and duration (D). Patient outcomes were correlated with neuromonitoring results. Intraoperative alert criteria were established on the basis of observed intraoperative changes. RESULTS: Three patients developed severe foot drop with a muscle weakness functional grade ranging from 0/5 to 3/5. Two patients developed a mild foot drop with functional grade 4/5. Twenty-three patients had preoperative weakness in an L5 distribution. One-hundred two patients who had neither preoperative nor postoperative neurological complications served as a control group. Amplitude significantly decreased in patients with a severe postoperative deficit (P = 0.005) as did AUC and duration (P < 0.05). Intraoperative alert criteria defined as a >65% decrease in AUC resulted in a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100%, 91.4%, 12%, and 100%, respectively. When defining an alert criteria as a >50% decrease in amplitude, sensitivity, specificity, PPV, and NPV were 100%, 87.9%, 8.8%, and 100%, respectively. CONCLUSION: Reduction of TCMEP waveform associated with postoperative severe foot drop can be detected during lumbar surgery. Other waveform parameters such as AUC may predict foot drop better than the amplitude. Additional examinations in larger samples of foot drops are needed to validate these alert threshold findings. LEVEL OF EVIDENCE: 4.

Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9.

Iron is vital for many homeostatic processes, and its liberation from ferritin nanocages occurs in the lysosome. Studies indicate that ferritin and its binding partner nuclear receptor coactivator-4 (NCOA4) are targeted to lysosomes by a form of selective autophagy. By using genome-scale functional screening, we identify an alternative lysosomal transport pathway for ferritin that requires FIP200, ATG9A, VPS34, and TAX1BP1 but lacks involvement of the ATG8 lipidation machinery that constitutes classical macroautophagy. TAX1BP1 binds directly to NCOA4 and is required for lysosomal trafficking of ferritin under basal and iron-depleted conditions. Under basal conditions ULK1/2-FIP200 controls ferritin turnover, but its deletion leads to TAX1BP1-dependent activation of TBK1 that regulates redistribution of ATG9A to the Golgi enabling continued trafficking of ferritin. Cells expressing an amyotrophic lateral sclerosis (ALS)-associated TBK1 allele are incapable of degrading ferritin suggesting a molecular mechanism that explains the presence of iron deposits in patient brain biopsies.

Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization.

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells.

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action.

Long-term Evaluation of Cervical Disc Arthroplasty with the Mobi-C© Cervical Disc: A Randomized, Prospective, Multicenter Clinical Trial with Seven-Year Follow-up.

BACKGROUND: Cervical total disc replacement (TDR) is an increasingly accepted procedure for the treatment of symptomatic cervical degenerative disc disease. Multiple Level I evidence clinical trials have established cervical TDR to be a safe and effective procedure in the short-term. The objective of this study is to provide a long-term assessment of TDR versus anterior discectomy and fusion for the treatment of one- and two-level disc disease. METHODS: This study was a continuation of a prospective, multicenter, randomized, US FDA IDE clinical trial comparing cervical TDR with the Mobi-C© Cervical Disc versus ACDF through 7 years follow-up. Inclusion criteria included a diagnosis of symptomatic cervical degenerative disc disease at one or two cervical levels. TDR patients were treated using a Mobi-C© artificial disc (Zimmer Biomet, Austin TX, USA). ACDF with allograft and anterior plate was used as a control treatment. Outcome measures were collected preoperatively and postoperatively at 6 weeks, at 3, 6, 12, 18 months, annually through 60 months, and at 84 months. Measured outcomes included Overall success, Neck Disability Index (NDI), VAS neck and arm pain, segmental range of motion (ROM), patient satisfaction, SF-12 MCS/PCS, major complications, and subsequent surgery rate. The primary endpoint was an FDA composite definition of success comprising clinical improvement and an absence of major complications and secondary surgery events. RESULTS: A total of 599 patients were enrolled and treated, with 164 treated with one-level TDR, 225 treated with two-level TDR, 81 treated with one-level ACDF, and 105 treated with two-level ACDF. At seven years, follow-up rates ranged from 73.5% to 84.4% (overall 80.2%).The overall success rates of two level TDR and ACDF patients were 60.8% and 34.2%, respectively (p<0.0001). The overall success rates of one level TDR and ACDF patients were 55.2% and 50%, respectively (p>0.05). Both the single and two level TDR and ACDF groups showed significant improvement from baseline NDI scores, VAS neck and arm pain scores, and SF-12 MCS/PCS scores (p<0.0001). In the single level cohort, there was an increased percentage of TDR patients who reported themselves as "very satisfied" (TDR 90.9% vs ACDF 77.8%; p= 0.028). There was a lower rate of adjacent level secondary surgery in the single level TDR patients (3.7%) versus the ACDF patients (13.6%; p = 0.007).In the two level TDR group, the NDI success rate was significantly greater in the TDR group (TDR: 79.0% vs. ACDF: 58.0%; p=0.001). There was significantly more improvement in NDI change score at 7 years in the TDR patients versus ACDF. The TDR group had a significantly higher rate of patients who were "very satisfied" with their treatment compared to the ACDF group (TDR: 85.9% vs. ACDF: 73.9%). The rate of subsequent surgery at the index level was significantly lower in the TDR group compared to the ACDF group (TDR: 4.4% vs. ACDF: 16.2%; p=0.001). The rate of adjacent level secondary surgery was significantly lower in the two level TDR (4.4%) patients compared to the ACDF (11.3%; p=0.03) patients. In both single and two level cohorts, the percentage of patients with worse NDI (2.5%-3.8% of two level surgeries and 1.2%-2.5% of single level surgeries) or worse neck pain (5%-6.8% of the two level surgeries and 1.3% - 3.8% of the single level surgeries) was strikingly low in both groups but trended lower in the TDR patients. CONCLUSIONS: At seven years, the composite success analysis demonstrated clinical superiority of two level TDR over ACDF and non-inferiority of single level TDR versus ACDF. There were lower rates of secondary surgery and higher adjacent level disc survivorship in both groups. Both surgeries were remarkably effective in alleviating pain relative to baseline and the rate of patients with worse disability or neck pain was surprisingly low. Overall, greater than 95% of patients (from both groups) who underwent TDR and 88% of patients who underwent ACDF were "very satisfied" at seven years. The differences in clinical effectiveness of TDR versus ACDF becomes more apparent as treatment increases from one to two levels, indicating a significant benefit for TDR over ACDF for two-level procedures. ETHICAL STANDARDS: The Mobi-C Clinical Trial (ClinicalTrials.gov registration number: NCT00389597) was conducted at 24 sites in the US and was approved by the Institutional Review Board, Research Ethics Committee, or local equivalent of each participating site. LEVEL OF EVIDENCE: 1.

DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks.

The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.

Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62.

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.

Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5.

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.

Subsequent surgery rates after cervical total disc replacement using a Mobi-C Cervical Disc Prosthesis versus anterior cervical discectomy and fusion: a prospective randomized clinical trial with 5-year follow-up.

OBJECTIVE Cervical total disc replacement (TDR) has been shown in a number of prospective clinical studies to be a viable treatment alternative to anterior cervical discectomy and fusion (ACDF) for the treatment of symptomatic degenerative disc disease. In addition to preserving motion, evidence suggests that cervical TDR may result in a lower incidence of subsequent surgical intervention than treatment with fusion. The goal of this study was to evaluate subsequent surgery rates up to 5 years in patients treated with TDR or ACDF at 1 or 2 contiguous levels between C-3 and C-7. METHODS This was a prospective, multicenter, randomized, unblinded clinical trial. Patients with symptomatic degenerative disc disease were enrolled to receive 1- or 2-level treatment with either TDR as the investigational device or ACDF as the control treatment. There were 260 patients in the 1-level study (179 TDR and 81 ACDF patients) and 339 patients in the 2-level study (234 TDR and 105 ACDF patients). RESULTS At 5 years, the occurrence of subsequent surgical intervention was significantly higher among ACDF patients for 1-level (TDR, 4.5% [8/179]; ACDF, 17.3% [14/81]; p = 0.0012) and 2-level (TDR, 7.3% [17/234]; ACDF, 21.0% [22/105], p = 0.0007) treatment. The TDR group demonstrated significantly fewer index- and adjacent-level subsequent surgeries in both the 1- and 2-level cohorts. CONCLUSIONS Five-year results showed treatment with cervical TDR to result in a significantly lower rate of subsequent surgical intervention than treatment with ACDF for both 1 and 2 levels of treatment. Clinical trial registration no.: NCT00389597 ( clinicaltrials.gov ).