CRISPR/Cas9 loss-of-function screen in a neuronal model of AP-4 deficiency identifies ATG9A trafficking modulators
Marvin Ziegler, Cedric Günter, Julian E. Alecu, Xutong Xue, Hyo M. Kim, Afshin Saffari, Alexandra K. Davies, Mustafa Sahin, Darius Ebrahimi-Fakhari
Marvin Ziegler, Cedric Günter, Julian E. Alecu, Xutong Xue, Hyo M. Kim, Afshin Saffari, Alexandra K. Davies, Mustafa Sahin, Darius Ebrahimi-Fakhari
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Research Article Cell biology Genetics Neuroscience

CRISPR/Cas9 loss-of-function screen in a neuronal model of AP-4 deficiency identifies ATG9A trafficking modulators

Abstract

Biallelic loss-of-function variants in adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and a degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the “druggable genome,” in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting that they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.

Authors

Marvin Ziegler, Cedric Günter, Julian E. Alecu, Xutong Xue, Hyo M. Kim, Afshin Saffari, Alexandra K. Davies, Mustafa Sahin, Darius Ebrahimi-Fakhari

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Figure 1

Establishment of a high-content CRISPR/Cas9 loss-of-function screening platform.

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Establishment of a high-content CRISPR/Cas9 loss-of-function screening p...
(A) Experimental workflow for CRISPR/Cas9 screen. Control and library sgRNAs were transferred into 384-well plates preloaded with Lipofectamine RNAiMAX. Differentiated Cas9-expressing SH-SY5Y cells were plated on top and incubated with sgRNAs for 72 hours. Cells were subsequently fixed and stained with Hoechst 33258 (nuclear marker), β-tubulin III (cytoplasmic marker), TGN46 (trans-Golgi network marker), and ATG9A (ATG9A compartment marker). Plates were imaged using an automated high-content imaging platform (Molecular Devices ImageXpress Micro Confocal Laser System and Peak Analysis & Automation GX robot arm), and images were analyzed using an automated pipeline in MetaXpress to segment cells and calculate the proportion of ATG9A signal overlapping with the TGN. This figure was partially created in BioRender (Kim H, 2026, https://BioRender.com/jrlyt8e). Cellular compartment masks were derived from MetaXpress. (B) Western blot analysis of whole-cell lysates from differentiated AP4B1KO and AP4B1WT SH-SY5Y cells. AP4B1KO cells showed loss of AP4B1 protein and reduced levels of AP4E1, indicating successful AP4B1 knockout and decreased AP-4 complex formation. ATG9A levels were increased in AP4B1KO cells. Both cell lines demonstrated stable expression of Cas9. (C) ATG9A ratio of untransfected AP4B1KO and AP4B1WT cells, based on all wells from the primary screen. AP4B1KO cells showed a significant increase in the ATG9A ratio, consistent with accumulation of ATG9A in the TGN. Statistical analysis: 2-sided Student’s t test, ***P < 0.001, Cohen’s d ≈ 2.74. (D) Representative images of AP4B1KO and AP4B1WT SH-SY5Y cells stained with antibodies against ATG9A and TGN46. AP4B1KO cells exhibited perinuclear accumulation of ATG9A overlapping with the TGN. Pseudocolored images depict grayscale intensity. Scale bars: 10 μm.