Parallel functional assessment of m 6 A sites in human endodermal differentiation with base editor screens

N 6 -methyladenosine (m 6 A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m 6 A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m 6 A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m 6 A sites to screen for m 6 A 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 m 6 A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m 6 A site on ADM . Our study provides a functional screening of m 6 A sites and paves the way for functional studies of m 6 A at individual m 6 A site level.