Parallel functional assessment of m⁶A sites in human endodermal differentiation with base editor screens

N⁶-methyladenosine (m⁶A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m⁶A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m⁶A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m⁶A sites to screen for m⁶A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m⁶A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m⁶A site on ADM. Our study provides a functional screening of m⁶A sites and paves the way for functional studies of m⁶A at individual m⁶A site level.