CRISPR/Cas9 Mediated GFP Knock‐in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy. Issue 11 (27th May 2018)

• Record Type: Journal Article
• Authors: Wu, Zhiqiang; Zhao, Jinlin; Qiu, Minghan; Mi, Zeyun; Meng, Maobin; Guo, Yu; Wang, Hui; Yuan, Zhiyong
• Is Part Of: Biotechnology journal. Volume 13:Issue 11(2018)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2018-05-27
• Subjects: autophagy -- CRISPR/Cas9 -- GFP‐LC3 -- knock in; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.201700674
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract : 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. Abstract : Ectopically expressed GFP‐LC3 is one of the most used reporters for monitoring autophagy, but it has limitations in accuracy. In this study, the authors constructed an endogenously expressed GFP‐LC3 reporter via CRISPR/Cas9 mediated knock‐in. This knock‐in GFP‐LC3 reporter is driven by the native promoter of LC3, expresses at endogenous level and shows improved sensitivity and signal‐to‐noise ratio in monitoring autophagy. Compared with the ectopically expressed one, this GFP‐LC3 reporter constructed here can be used to accurately identify and quantify cellular autophagy.