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.