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

Tianwei Liu,Weisheng Cheng,Yixin Ma,Zhijun Ren,Wenfang Chen,Yaxin Chen,Fang Liu,Nan Cao,Jinkai Wang

doi:10.1038/s41467-022-28106-0

Abstract

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.

Highlights

N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells
We found that FNLS-BE3 and FNLSABE7.10(AW) nearly completely substituted their targeted nucleotides on HEK4 and METTL3-1 locus, respectively (Supplementary Fig. 1d, h)
We demonstrate that adenine base editors (ABEs) base editor can be used to functionally access tens of thousands of m6A sites in a pooled screening at base resolution

Summary

Introduction

N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. We and others previously found that depletion of the m6A methyltransferase complex results in blocked differentiation in both human and mouse embryonic stem cells (hESCs and mESCs)[8,9], illuminating that m6A methylation, which serves as a timely maintainer of the balance between pluripotency and lineage priming factors, is crucial in regulating cellular specification during embryogenesis. These pioneer studies have shown that m6A in mRNA may work as a ‘plug-in’ to other pre-existing pathways by altering downstream gene expression. The cytosine base editors (CBEs) use the rat cytidine deaminase enzymes

Methods

Results

Conclusion