Generation of CRISPR-Cas9 ATG7 and ATG9 knockout cell lines

To generate knock out cell lines of autophagy-related genes, we utilized the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system [42]. We designed RNA-guided Cas9 targeting the first exons of the human ATG7 and ATG9 genes. The specific recognition sequences of the 20 bp before the protospacer adjacent motif (PAM) in each construct were as follows: ATG7, 5’- CACCGAAATAATGGCGGCAGCTACG-3’; ATG9, 5’-CACCCCCTGGGGGTGAATCAC TAT-3’. These guide oligos, with BbsI restriction sites at both ends, were annealed with their anti-sense oligos and inserted downstream of U6 promoter in vector pSpCas9(BB)-2A-GFP (pX458) [43] (purchased from Addgene); the resultant plasmids were used for transfection. Single-cell sorting was performed after a 48-h transfection. After 1-week culture of single cells with antibiotics, fresh medium was added to support growth for 1 more week to allow colony formation. Genomic DNA of each clone was extracted, and the target gene was confirmed by sequencing using the following primers; ATG7, Fw 5’-GTCGACGTTCTGGAGATCTGTTTCACAACG-3’ and Re 5’-GAATTCTGGGATCAAAAAGTCAGGAAG-3’; ATG9, Fw 5’-ATATGTCGACCAGGATGAGCTCCATTCCCGT-3’ and Re 5’-ATATGAATTCCAGCCCCCAACAAAGGGACAG-3’.