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CRISPR/Cas9 Mediated GFP Knock‐in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy
Author(s) -
Wu Zhiqiang,
Zhao Jinlin,
Qiu Minghan,
Mi Zeyun,
Meng Maobin,
Guo Yu,
Wang Hui,
Yuan Zhiyong
Publication year - 2018
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700674
Subject(s) - autophagy , green fluorescent protein , crispr , microbiology and biotechnology , cas9 , endogeny , biology , gene knockin , reporter gene , genetics , gene , gene expression , apoptosis , biochemistry
Accurately identifying and quantifying cellular autophagy is very important as the significance of autophagy in physiological and pathological processes becomes increasingly evident. Ectopically expressed fluorescent‐tagged microtubule‐associated protein light chain 3B ( MAP1LC3B, LC3 ) is the most widely used reporter for monitoring autophagy activity thus far. However, this approach ignores the influence of constitutively overexpressed LC3 on autophagy itself and autophagy‐related processes and its accuracy in indicating autophagy is questionable. Here, we generated a knock‐in GFP‐LC3 reporter via the CRISPR/Cas9 system in 293FT cells to add GFP to the N‐terminal of and in frame with endogenous LC3. We proved that this knock‐in GFP‐LC3 was expressed at biological level driven by the endogenous transcriptional regulatory elements as the wild type alleles. Compared with the ectopically expressed GFP‐LC3, the endogenous knock‐in reporter exhibited much higher sensitivity and signal‐to‐noise ratio of GFP‐LC3 puncta upon the induction or inhibition of autophagy at certain step for monitoring autophagy activity. Thus, according to the previous reported concerning and the results presented here, we suggest that this knock‐in GFP‐LC3 reporter is better for bona fide monitoring cellular autophagy and should be employed for further study of autophagy in vitro and in vivo.