Whole genome CRISPR knockout screen to identify targets for cardiac differentiation and disease

Inherited cardiac diseases arise from single or multiple mutations in cardiac genes and can lead to severe conditions such as hypertrophic cardiomyopathy or arrhythmias. However, developmental mechanisms are difficult to study because animal models often fail to accurately reflect human cardiac biology. To address this, we used our double human embryonic stem cell (hESC) atrial and ventricular reporter NKX2.5EGFP/+-COUP-TFII-mCherry/+ cell line which enables selection of both atrial and ventricular differentiated cardiomyocytes. We further modified this into a Cas9-expressing hESC line suitable for large-scale knockout screenings and specific gene targeting. State-of-the-art CRISPR/Cas9 loss-of-function screens have proven effective for uncovering regulators of essential biological pathways. Using this approach, we aim to identify genes essential during human cardiac development for atrial and ventricular specification, and potential drivers of cardiac disease. We used a dual-knockout CRISPR library containing ~91,000 sgRNAs to cover the whole human genome with ~21,000 genes and performed the screen at >200× coverage at the single-cell level. These cells, each containing a single knockout, were then subjected to negative selection by differentiating into atrial and ventricular cardiomyocytes. Following differentiation, next-generation sequencing and MaGeCK analysis were used to quantify sgRNA representation and identify essential targets. In this study, we carried out a genome-wide CRISPR-Cas9 loss-of-function screen in hESCs to uncover genes that specifically influence cardiac differentiation and contribute to the molecular basis of inherited cardiac disease.