Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

Kailong Li,Yuxuan Liu,Pranita Kaphle,Andy Yu,Jian Xu,Min Ni,Hui Cao,Xin Liu,Yuannyu Zhang,Zhimin Gu,Kathryn E Dickerson

doi:10.1038/s41467-020-14362-5

Abstract

Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe CRISPR/dCas9-based enhancer-targeting epigenetic editing systems, enCRISPRa and enCRISPRi, for efficient analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the improved systems display more robust perturbations of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Single or multi-loci perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoiesis. Hence, enhancer-targeting CRISPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.

Highlights

Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cisregulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging
Since H3K27ac is the hallmark of active enhancers[33], by doxycycline (Dox)-inducible expression of deactivated Cas9 (dCas9)-p300, sgRNA-MS2 and MCP-VP64, we engineered the enhancertargeting dual-activator systems
Using epigenetic writer proteins (p300 and LSD1) that modulate histone modifications associated with active enhancers, the enCRISPRa and enCRISPRi systems enable efficient interrogation of enhancer function in human and mouse cells in vitro, in xenografts and in vivo

Summary

Introduction

Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cisregulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Systematic annotation of human epigenomes has identified millions of putative CREs using correlative features such as chromatin accessibility and histone modifications[1,2,3]; the analysis of in vivo functions of these elements within their native chromatin remains difficult This is in part because existing technologies often measure enhancer activity in heterologous assays without native chromatin, and because findings from these assays have not been causally connected with specific target genes or cellular functions during development. Mediated transcriptional activity at enhancer DNA sequences, but they have some important limitations including the lack of local chromatin contexts and epigenetic features in heterologous assays, the often use of a general promoter such as SV40 rather than the enhancer’s endogenous promoter, the inability to identify the target genes of enhancers, and the inadequacy to model combinatorial regulation by multiple enhancers at native chromatin. High-resolution saturating mutagenesis of cis-regulatory elements relies on loss-offunction and does not permit gain-of-function analyses[13,14]

Methods

Results

Conclusion