小柯机器人

日本京都大学Itaru Hamachi和Tomonori Tamura团队合作的研究表明，细胞器选择性点击标记与流式细胞术相结合可利用CRISPR库对磷脂酰胆碱代谢基因进行筛选。相关论文发表在2023年3月13日出版的《细胞-代谢》杂志上。

研究人员将磷脂酰胆碱（PC）的细胞器选择性点击标记与基于流式细胞术的CRISPR筛选相结合，将细胞器PC表型转换为可用于全基因组筛选的简单荧光读数。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，以鉴定已报道的与PC合成（PCYT1A、ACACA）、囊泡膜运输（SEC23B、RAB5C）和非囊泡运输（PITPNB、STARD7）相关的基因。

此外，研究人员还发现FLVCR1是胆碱摄取的促进剂，CHEK1是PC合成途径的翻译后调节因子以及先前未发现的CDC50A功能-参与调控PC易位到质膜双层外部。这些发现证明了O-ClickFC平台在细胞脂质代谢遗传解析方面的多功能性。

据了解，细胞脂质的合成和转运是由复杂的蛋白质网络控制。尽管基因筛查有助于破解脂质代谢的调控网络，但在技术上仍具有挑战性，特别是对脂质表型进行高通量筛选。

附：英文原文

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

Author: Masaki Tsuchiya, Nobuhiko Tachibana, Kohjiro Nagao, Tomonori Tamura, Itaru Hamachi

Issue&Volume: 2023-03-13

Abstract: Cellular lipid synthesis and transport are governed by intricate protein networks.Although genetic screening should contribute to deciphering the regulatory networksof lipid metabolism, technical challenges remain—especially for high-throughput readoutsof lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine(PC) with flow cytometry-based CRISPR screening technologies to convert organellarPC phenotypes into a simple fluorescence readout for genome-wide screening. This technique,named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screensto 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 membranebilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedentedplatform for genetic dissection of cellular lipid metabolism.

DOI: 10.1016/j.cmet.2023.02.014

Source: https://www.cell.com/cell-metabolism/fulltext/S1550-4131(23)00050-5

Cell Metabolism：《细胞—代谢》，创刊于2005年。隶属于细胞出版社，最新IF：31.373
官方网址：https://www.cell.com/cell-metabolism/home
投稿链接：https://www.editorialmanager.com/cell-metabolism/default.aspx