Stable CRISPR-Cas9 HEK293T Cell Line

A HEK293T cell line stably expressing GFP-LC3-RFP and Cas9 (HEK293T GFP-LC3-RFP cells) was generated as previously described (6 (link)). LentiCas9-Blast (52962 (37 (link)); Addgene), human GeCKO v2 Library (1000000049 (37 (link)); Addgene), lentiCRISPR v2 (52961 (37 (link)); Addgene), and pSp-Cas9(BB)-2A-Puro (48139 (64 (link)); Addgene) were provided by F. Zhang (Massachusetts Institute of Technology, Cambridge, MA). For CRISPR knockouts, sgRNAs were cloned into lentiCRISPR v2 and pSp-Cas9(BB)-2A-Puro, sequences of which are listed in Table S5. For stable expression, GFP-LC3-RFP reporter was inserted into pMRX-IP (65 (link)) and pMRX-IB (6 (link)), and PFAS (NM_012393) tagged with 3 × FLAG was inserted into pMRX-IB (6 (link)) (these plasmids were generated from pMXs (66 (link))).

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Mimura K., Sakamaki J.I., Morishita H., Kawazu M., Mano H, & Mizushima N. (2021). Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy. The Journal of Biological Chemistry, 296, 100780.

Publication 2021

LentiCas9-Blast human GeCKO v2 Library lentiCRISPR v2 pSp-Cas9(BB)-2A-Puro

Cell line Cells Crispr Gecko Human Library Plasmids

Corresponding Organization : The University of Tokyo

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Variable analysis

independent variables

CRISPR knockouts using sgRNAs cloned into lentiCRISPR v2 and pSp-Cas9(BB)-2A-Puro
Stable expression of GFP-LC3-RFP reporter inserted into pMRX-IP and pMRX-IB
Stable expression of PFAS (NM_012393) tagged with 3 × FLAG inserted into pMRX-IB

dependent variables

Not explicitly mentioned

control variables

HEK293T cell line stably expressing GFP-LC3-RFP and Cas9 (HEK293T GFP-LC3-RFP cells)

controls

Positive control: Not specified
Negative control: Not specified

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