CRISPR-Cas9–mediated knockout was performed using an established methodology (38). The short guide RNA (sgRNA) sequence (5′-AAATTTGTCAGCAACTCGTC-3′) from the GeCKO library (39) was cloned into the Bbs I restriction site of the SpCas9-2A-Puro V2.0 vector, which was a gift from F. Zhang (Addgene plasmid no. 62988). MDA-MB-231 cells were transiently transfected with the sgRNA plasmid, grown for 48 hours, and selected with puromycin (0.5 μg/ml; Invitrogen). Surviving cells were plated as single-cell colonies in a 96-well plate (1 cell per well) and expanded. Knockout of TRPM7 in expanded cells was confirmed by Western blotting with an anti-TRPM7 antibody (clone N74/25, Abcam).

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