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Resource and Technical AdvanceIn-Press PreviewCell biologyGeneticsNeuroscience Open Access | 10.1172/jci.insight.202204

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

Marvin Ziegler,1 Cedric Günter,1 Julian E. Alecu,1 Xutong Xue,1 Hyo-Min Kim,1 Afshin Saffari,1 Alexandra K. Davies,2 Mustafa Sahin,1 and Darius Ebrahimi-Fakhari1

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Ziegler, M. in: PubMed | Google Scholar |

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Günter, C. in: PubMed | Google Scholar

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Alecu, J. in: PubMed | Google Scholar

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Xue, X. in: PubMed | Google Scholar

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Kim, H. in: PubMed | Google Scholar

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Saffari, A. in: PubMed | Google Scholar

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Davies, A. in: PubMed | Google Scholar

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Sahin, M. in: PubMed | Google Scholar |

1Department of Neurology and F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard Medical School, Boston, United States of America

2School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, United Kingdom

Find articles by Ebrahimi-Fakhari, D. in: PubMed | Google Scholar |

Published May 19, 2026 - More info

JCI Insight. https://doi.org/10.1172/jci.insight.202204.
Copyright © 2026, Ziegler et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Published May 19, 2026 - Version history
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Abstract

Biallelic loss-of-function variants in the adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including the 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 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 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.

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Version history
  • Version 1 (May 19, 2026): In-Press Preview
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