Interrogation of Enhancer Function by Enhanced CRISPR Epigenetic Editing

Abstract

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. 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 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 new systems display more robust perturbation 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. Multiplexed perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoietic lineage specification. Hence, enhanced CRSIPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts. 16 weeks Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting: Controls Enhancers Promoters sgRNAs targeting:

Detection of gene cis-regulatory element perturbations in single-cell transcriptomes

“…However, CRISPRi is limited in resolution as it typically makes use of a KRAB repressor domain tethered to a nuclease-inactivated Cas9 to induce heterochromatin over 1-2kb. Furthermore, the KRAB repressor domain may not accurately perturb enhancer function at distal sites [ 26 ]. This is in contrast to CRISPR mutational screens, which depend on error-prone repair following CRISPR/Cas9-induced double-stranded breaks.…”