Abstract: N6-methyladenosine (m6A) modification represents a crucial epitranscriptomic regulatory mechanism in stem cell fate determination. However, its role in human neural development remains largely unexplored. Here, we first systematically profiled m6A modifications during human embryonic stem cell (hESC) differentiation into three germ layers, revealing dynamic m6A patterns associated with lineage specification. Through CRISPR-mediated knockout of METTL3, the catalytic subunit of m6A methyltransferase complex, we identified extensive transcriptional changes during germ layer differentiation, with particularly profound effects on ectodermal lineage commitment. Further investigation using hESC-derived brain organoids demonstrated that METTL3 depletion resulted in severe developmental defects, particularly characterized by reduced organoid size and decreased expression of cortical neuron markers TBR1 and CTIP2. Single-cell transcriptome profiling of day 60 organoids revealed distinct alterations in cellular composition and neuronal differentiation trajectories upon METTL3 deletion. Our findings demonstrate that METTL3-mediated m6A modification is indispensable for human neural development and provide new insights into the epitranscriptomic regulation of brain development.
