2. Academic Validation
  3. Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism

Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism

  • Cell Metab. 2023 Jun 6;35(6):1072-1083.e9. doi: 10.1016/j.cmet.2023.02.014.
Masaki Tsuchiya 1,  Nobuhiko Tachibana 1,  Kohjiro Nagao 2,  Tomonori Tamura 3,  Itaru Hamachi 4
Affiliations

Affiliations

  • 1 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; PRESTO (Precursory Research for Embryonic Science and Technology), JST, Sanbancho, Chiyodaku, Tokyo 102-0075, Japan.
  • 2 Department of Biophysical Chemistry, Kyoto Pharmaceutical University, 5 Misasaginakauchi-cho, Yamashina-ku, Kyoto 607-8414, Japan.
  • 3 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. Electronic address: [email protected].
  • 4 Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; ERATO (Exploratory Research for Advanced Technology), JST, Sanbancho, Chiyodaku, Tokyo 102-0075, Japan. Electronic address: [email protected].
PMID: 36917984 DOI: 10.1016/j.cmet.2023.02.014
Abstract

Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.

Keywords

CRISPR screens; flow cytometry; lipid metabolism; organelle-selective labeling: click chemistry; phosphatidylcholine.

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