Generation of the AMPK α1 knockout cell line with CRISPR/Cas9

Guide RNA sequences for use in the CRISPR/Cas9 system were designed at the CRISPR design website (http://crispr.mit.edu/), provided by the Feng Zhang Lab²⁴. The sequences for the insert oligonucleotides for human AMPK α1 gRNA #1 and #2 were as follows 5′-CACCGA AGATCGGCCACTACATTC-3′/5′-AAACGAATGTAGTGGCCGATCTTC-3′and 5′-CACCGCCGAGAAGCAGAAACACGA-3′/5′-AAACTCGTGTTTCTGCTTCTCGGC-3′, respectively. The AMPK α1 guide RNA targets exon 1 of the AMPK α1 gene. The complementary oligonucleotides for the guide RNAs (gRNAs) were cloned into a pX459 CRISPR/Cas9-Puro vector (Addgene, Cambridge, MA, USA). HEK293T cells were transfected with lipofectamine transfection reagent (Invitrogen, Carlsbad, CA, USA). Two days after transfection, cells were treated with 1 μg/ml of puromycin (Sigma) for three days. After two weeks, colonies were isolated, and the AMPK α1 sequences were amplified by PCR and analyzed using the T7 endonuclease (T7E1) assay, DNA sequencing (Macrogen, Seoul, Korea), and Western blotting. The T7E1 enzyme was purchased from New England Biolabs (Beverly, MA, USA).