Pooled CRISPR screening identifies m⁶A as a positive regulator of macrophage activation

m⁶A RNA modification is implicated in multiple cellular responses. However, its function in the innate immune cells is poorly understood. Here, we identified major m⁶A "writers" as the top candidate genes regulating macrophage activation by LPS in an RNA binding protein focused CRISPR screening. We have confirmed that Mettl3-deficient macrophages exhibited reduced TNF-α production upon LPS stimulation in vitro. Consistently, Mettl3 ^flox/flox;Lyzm-Cre mice displayed increased susceptibility to bacterial infection and showed faster tumor growth. Mechanistically, the transcripts of the Irakm gene encoding a negative regulator of TLR4 signaling were highly decorated by m⁶A modification. METTL3 deficiency led to the loss of m⁶A modification on Irakm mRNA and slowed down its degradation, resulting in a higher level of IRAKM, which ultimately suppressed TLR signaling-mediated macrophage activation. Our findings demonstrate a previously unknown role for METTL3-mediated m⁶A modification in innate immune responses and implicate the m⁶A machinery as a potential cancer immunotherapy target.