CRISPR/Cas9 m6A metabolic enzyme knockout screening

Two types of circular RNAs (circRNAs) incorporated split GFP along with an m6A motif were performed following previous protocol30,31. CRISPR-Cas9 knockout library contained 7142 sgRNAs targeting for 1773 classical metabolic enzyme genes, with four designed sgRNAs per gene. The m6A GFP reporter gene plasmid was packaged into lentivirus, and the obtained virus was used to infect HEK293T cells. After 48 hours, puromycin selection was performed. To establish a homogeneous fluorescent cell line for subsequent experiments, single clones were selected. CRISPR-Cas9-mediated knockout of metabolic enzyme-related genes was carried out in HEK293T single clone cells infected with the lentiviral library. The transfection efficiency was maintained around 30%. Seven days after viral addition, flow cytometry was conducted following puromycin selection to isolate cells exhibiting the weakest fluorescence intensity (5%). Genomic DNA was extracted from these cells, libraries were constructed, and subsequent sequencing analysis was performed. The sgRNA read count and calling of hits were analyzed using MAGeCK v0.5.6.