ChIP-seq analysis of cas9, HA, H3K4me, H3K4me2, H3K9me3, H3K27ac, CTCF, GATA1 and TAL1 in HS2 enhancer perturbed K562 cells or other leukemia cell lines

Tissue-specific gene expression requires coordinated control of gene-proximal and distal cis-regulatory elements (CREs), yet functional analysis of putative gene-distal CREs such as enhancers remains challenging. Here we describe enhanced CRISPR/dCas9-based epigenetic editing systems, enCRISPRa and enCRISPRi, for multiplexed analysis of enhancer function in situ and in vivo. Using dual effector proteins 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, our systems display more robust and specific perturbations of gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Furthermore, multiplexed perturbations of lineage-specific enhancers in an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted requirement of developmentally regulated enhancers during hematopoietic lineage specification. Hence, enhanced CRSIPR epigenetic editing provides opportunities for interrogating enhancer function in development and disease. Overall design: ChIP-seq was performed to determine the chromatin occupancy of Cas9, HA, H3K4me, H3K4me2, H3K9me3, H3K27ac, H3K27me3, CTCF, GATA1 and TAL1 in HS2 enhancer perturbed K562 cells or other leukemia cell lines