AMPK-deficient human T cells decrease oxidative and glycolytic metabolism in vitro. CD3⁺ human T cells were purified from the peripheral blood of healthy human donors, electroporated with ribonucleoprotein (RNP) complexes containing the Cas9 protein and a guide RNA (gRNA) targeting human AMPKα1 locus and expanded until day 10 in recombinant human IL-2. (A) Genomic DNA purified from day 10 T cells was used to amplify a 700 bp fragment covering the gRNA target site, followed by Sanger sequencing and decomposition analysis.¹⁹ (B) Protein from 1 × 10⁵ day 10 T cells was precipitated with trichloroacetic acid (TCA) followed by immunoblot analysis for specific phosphorylation of ULK-1 on Ser555 by AMPK. (C-H) Day 10 T cells were stimulated overnight with CD3/CD28 dynabeads in physiologic glucose (5.5 mM) and placed on the Seahorse metabolic analyzer (C). Both maximal OCR (D) and SRC (E) were measured simultaneously with ECAR (F-H), including both the response to oligomycin (G) and the maximal ECAR values (H). Plots in panels C,F are representative of 2 independent donors, whereas data in panels D-G are the composite analysis from these 2 donors. ∗∗∗P < .001; ∗∗∗∗P < .0001.