Fig. 1: High-throughput in vivo CRISPR–Cas9-based forward genetic screen for regulators of cardiomyocyte maturation.
a Schematic overview of the screen design. On-chip oligonucleotide synthesis was used to generate a pooled AAV library containing ~15 k gRNAs that targeted transcriptional regulators. These were delivered to neonatal mice, and a flow cytometry (FACS)-based single-cell screening assay was used to identify immature CMs. Enriched and depleted guides were identified by next-generation sequencing (NGS) of gRNAs. Artwork from biorender.com. b Myh7YFP expression is restricted to neonatal stage. Scale bars = 5 μm; n = 5 mice. c Depletion of both GATA4 and GATA6 by CASAAV resulted in persistent MYH7YFP expression. Data are presented as mean values + /− SEM. n = 3 mice per group. d Sample clustering by Pearson correlation (r). e DESeq2 differential expression analysis of individual gRNAs. Red dots are gRNAs with Benjamini–Hochberg corrected Wald test p-value < 0.001. GATA4 P = 1.5 × 10−16, GATA6 P = 2.4 × 10−21. f Enrichment of positive (GATA4/6) and negative control gRNAs within YFP+ CMs as calculated by DESeq2. g Gene depletion as calculated by MAGeCK. h Gene enrichment as calculated by MAGeCK.
