To study the role of mammalian Atg8-family (mATG8) proteins in autophagosome biogenesis, we employed the CRISPR-Cas9 genome editing system to generate triple-knockouts (TKOs) of LC3 (LC3A, LC3B, LC3), GBR (GBR, GBRL1, GBRL2), and both subfamilies (hexa-knockout, HKO) in U-2 OS human osteosarcoma cells. Here, MAP1LC3B is linked to osteosarcoma.