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
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DOI：
10.1016/j.cmet.2023.02.014
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发表时间：
2023-06-06
期刊：
影响因子：
29
通讯作者：
Hamachi, Itaru
中科院分区：
文献类型：
--
作者：
Tsuchiya, Masaki;Tachibana, Nobuhiko;Hamachi, Itaru
细胞脂质的合成和运输受复杂的蛋白质网络控制。虽然遗传筛查有助于破译脂质代谢的调控网络，但技术挑战仍然存在-特别是对于脂质表型的高通量读数。在这里，我们将磷脂酰胆碱（PC）的细胞器选择性点击标记与基于流式细胞术的CRISPR筛选技术相结合，将细胞器PC表型转换为简单的荧光读数，用于全基因组筛选。这种名为O-ClickFC的技术已成功应用于基因组规模的CRISPR敲除筛选，以识别先前报道的与PC合成（PCYT 1A，ACACA），囊泡膜运输（SEC 23 B，RAB 5C）和非囊泡运输（PITPNB，STARD 7）相关的基因。此外，我们揭示了FLVCR 1作为胆碱摄取促进剂，CHEK 1作为PC合成途径的翻译后调节剂，以及CDC 50 A作为负责PC易位到质膜双层外的先前未知的角色。这些发现证明了O-ClickFC作为细胞脂质代谢遗传解剖的前所未有的平台的多功能性。
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 label-ing 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 previ-ously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously unchar-acterized 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.