Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism
Tsuchiya, M.; Tachibana, N.; Nagao, K.; Tamura, T.; Hamachi, I.
Show abstract
Lipids comprise biomembranes and are involved in many crucial cell functions. While cellular lipid synthesis and transport appear to be governed by intricate protein networks, the whole scheme is insufficiently understood. Although functional genome-wide 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, this work revealed previously uncharacterized roles of FLVCR1 as a new choline transporter; CHEK1 as a post-translational regulator of the PC-synthetic pathway, and TMEM30A as responsible for 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.
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