Establishment of a highly efficient gene disruption strategy to analyze and manipulate lipid co-regulatory networks
Harayama, T.; Hashidate-Yoshida, T.; Aguilera-Romero, A.; Hamano, F.; Morimoto, R.; Shimizu, T.; Riezman, H.
Show abstract
Gene disruption has been dramatically facilitated by genome editing tools. Despite improvements in gene disruption rates in cultured cells, clone isolation remains routinely performed to obtain mutants, potentially leading to artifacts due to clonal variation in cellular phenotypes. Here we report GENF, a highly efficient strategy to disrupt genes without isolating clones, which can be multiplexed. Using it, we obtained reliable lipidomics datasets from mutant cells without being affected by variances related to clone isolation. Through this, we found that an enzyme involved in congenital generalized lipodystrophy regulates glycerophospholipids with specific acyl-chains. We also demonstrate the possibility to dissect complex lipid co-regulatory mechanisms, explaining cell adaptations to altered lipid metabolism. With its simplicity and the avoidance of cloning-related artifacts, GENF is likely to contribute to many cell biology studies, especially those involving -omics approaches.
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