This activity of ZBTB16 was alternatively tested by acutely knocking out the gene in the human monocytic THP-1 cell line using DOX-inducible CRISPR gene editing. The phenotype of these cells was validated by the detection of fluorescent reporters for Cas9 and guide RNA expression, measures of the ZBTB16 transcript and rescue of the DOX-induced deficiency by exogenous re-expression of ZBTB16 (Supplementary Fig. 2c–e). Transiently decreasing ZBTB16 in these cells reduced IL-1β release and cytotoxicity in response to treatment with LPS and nigericin or by treatment with C. difficile or its purified TcdB toxin (Fig. 2g).
