CRISPR/Cas9 Mediated GFP Knock‐in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy,Biotechnology Journal

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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
Biotechnology Journal ( IF 3.2 ) Pub Date : 2018-05-27 , DOI: 10.1002/biot.201700674
Zhiqiang Wu ₁ , Jinlin Zhao ₁ , Minghan Qiu ₁ , Zeyun Mi ₂ , Maobin Meng ₁ , Yu Guo ₃ , Hui Wang ₁ , Zhiyong Yuan ₁

Affiliation

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.

中文翻译：

CRISPR / Cas9介导的293FT细胞中MAP1LC3B基因座上的GFP敲入更好地用于善意监测细胞自噬

随着细胞自噬在生理和病理过程中的重要性日益明显，准确鉴定和定量细胞自噬非常重要。异位表达荧光标记的微管相关蛋白轻链3B（MAP1LC3B，LC3）是迄今为止监测自噬活性最广泛使用的报告基因。但是，这种方法忽略了组成型过表达的LC3对自噬本身和与自噬相关的过程的影响，其在指示自噬方面的准确性值得怀疑。在这里，我们通过CRISPR / Cas9系统在293FT细胞中生成了敲入的GFP-LC3报告基因，以将GFP添加到内源LC3的N端并与之符合读框。我们证明了这种敲入的GFP-LC3在由野生型等位基因在内源转录调节元件驱动的生物学水平上表达。与异位表达的GFP-LC3相比，内源性敲入报告基因在监测或监测自噬活性的特定步骤中自噬被诱导后表现出更高的GFP-LC3小点敏感性和信噪比。因此，

更新日期：2018-09-17

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