CHD-associated enhancers shape human cardiomyocyte lineage commitment

Abstract

Enhancers orchestrate gene expression programs that drive multicellular development and lineage commitment. Thus, genetic variants at enhancers are thought to contribute to developmental diseases by altering cell fate commitment. However, while many variant-containing enhancers have been identified, studies to endogenously test the impact of these enhancers on lineage commitment have been lacking. We perform a single-cell CRISPRi screen to assess the endogenous roles of 25 enhancers and putative cardiac target genes implicated in genetic studies of congenital heart defects (CHD). We identify 16 enhancers whose repression leads to deficient differentiation of human cardiomyocytes (CMs). A focused CRISPRi validation screen shows that repression of TBX5 enhancers delays the transcriptional switch from mid- to late-stage CM states. Endogenous genetic deletions of two TBX5 enhancers phenocopy epigenetic perturbations. Together, these results identify critical enhancers of cardiac development and suggest that misregulation of these enhancers could contribute to cardiac defects in human patients.

CRISPR Screening Uncovers a Long-Range Enhancer forONECUT1in Pancreatic Differentiation and Links a Diabetes Risk Variant

“…We and others have applied CRISPR-Cas9 in hPSCs to uncover genes important for gastrulation and pancreatic development [17][18][19][20][21][22] . Recent CRISPRi screens have further identified enhancers involved in hPSC endoderm and cardiac differentiation 8,23,24 . These advances motivated us to leverage our epigenetic characterization of the hPSC pancreatic differentiation system 25 to uncover enhancers with potential roles in diabetes.…”

Technological Convergence: Highlighting the Power of CRISPR Single-Cell Perturbation Toolkit for Functional Interrogation of Enhancers

“…They functionally interrogated 25 enhancers associated with congenital heart defects in stem cells, ahead of their differentiation to cardiomyocytes. They measured the transcriptome and found a subset of 16 enhancers where the perturbation caused a delay in the specification of human cardiomyocytes [35]. In another study, a multi-assay evaluation of GWAS target genes was carried out using functional CRISPR screens, Highthroughput Chromosome Conformation Capture with Chromatin Immunoprecipitation (HiChIP), and crisprQTL to evaluate the relationship between candidate breast cancerassociated enhancers and their target genes [36].…”

“…In the context of CRISPR multiplexing perturbations, the competition of several sgRNAs for endonucleases in a cell, known as retroactive effects, can change the efficacy of every sgRNA [62]. A focused validation, through targeting enhancers by an active Cas9 protein, can also help to balance speed with accuracy [35].…”

“…Other factors, such as the level of activation, genomic locus, and neighboring elements like insulators, enhancers, and promoters can also influence this spreading [63]. Considering the time dependency of enhancer functions, a delay in the KRAB function could pose a technical challenge while sgRNAs are introduced at a fixed time point [35].…”