(A) Genome browser snapshot emphasizing features of a targeted enhancer about 15 kb upstream of the RBM20 gene. Yellow highlights enhancer region. (B) H3K27ac and open chromatin (ATAC-Seq) …
(A) Brightfield images of H9-dCas9-KRAB cells receiving sgRNAs targeting OCT4 or NT control (Scale bar = 200μm). (B) (Top) OCT4 expression in cells receiving sgRNAs targeting OCT4 or NT control. For …
(A) PHATE visualization of CM cells with four Louvain clusters. (B) Feature plot of pseudotime ranking of cells across PHATE trajectory. (C) Feature plot of TNNT2 expression. We defined the top 100 …
(A) Feature plots of marker genes for SOX4+ progenitors, FN1+ early-stage CMs, ACTA2+ mid-stage CMs, and NPPA+ atrial-like late-stage CMs. (B) Example of sgRNA filtering approach. (Top) p-Values …
(A) (Top) Schematic of validation CRISPRi screen. H9-dCas9-KRAB cells are infected with lentiviral sgRNA library targeting TBX5 enhancers and differentiated over 8 days into CMs followed by …
(A) Feature plots of marker genes for neuronal (+SOX2), cardiomyocytes (+TNNT2), endoderm (+TTR). (B) Feature plots of cells receiving sgRNAs targeting TBX5 enhancers (red) or non-genome targeting …
PHATE visualization of CMs in the focused screen with three Louvain clusters. (B) Feature plot of pseudotime ranking of cells across PHATE trajectory. (C) Feature plot of NPPA expression. (D) …
(A) Feature plots of marker genes for CM progenitors (FN1+), mid-CM (ACTA2+), and late-CM (NPPA+). (B) Feature plots of cells receiving sgRNAs targeting TBX5 enhancers (red) or non-genome targeting …
(A) (Top) TBX5 enhancer knockout strategy. (Bottom) Genome browser snapshots of chromatin status and sequence conservation. Dotted lines denote sgRNA target sites. (B) Genotyping PCR to verify TBX5 …
Original genotyping gels for panel B.
Enhancer 3 KO genotyping gel, with labels.
Original genotyping gels for panel B.
Enhancer 3 KO genotyping gel, without labels.
Original genotyping gels for panel B.
Enhancer 5 KO genotyping gel, with labels.
Original genotyping gels for panel B.
Enhancer 5 KO genotyping gel, without labels.
Original genotyping gels for panel B.
TBX5 exon 3 KO genotyping gel, with labels.
Original genotyping gels for panel B.
TBX5 exon 3 KO genotyping gel, without labels.
Original western blots for panel N. Western blot of actin control, with labels.
Original western blots for panel N. Western blot of actin control, without labels.
Original western blots for panel N. Western blot of FN1, with labels.
Original western blots for panel N. Western blot of FN1, without labels.
(A) UMAP visualization of H9-derived cells after 8 days of differentiation. Five Louvain clusters indicated. (B) UMAP distribution of TBX5 exon KO and enhancer KO cells after cardiomyocyte (CM) …
(A) TBX5 enhancer 5 heterozygous KO strategy. (B) (Top) Position of primers used to validate TBX5 enhancer 5 heterozygous clone. Red: KO enhancer-spanning primers; blue: left junction primers; …
Original genotyping gels for panel B. Enhancer 5 HET genotyping, with labels.
Original genotyping gels for panel B. Enhancer 5 HET genotyping, without labels.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Chemical compound, drug | Puromycin | Cayman Chemical | Cat#13884 | |
Chemical compound, drug | Blasticidin | RPI | Cat#3513-03-9 | |
Chemical compound, drug | Thiazovivin | Sigma-Aldrich | Cat#SML1045 | |
Chemical compound, drug | TrypLE Select | Thermo Fisher | Cat#12563 | |
Chemical compound, drug | CHIR99021 | Tocris | Cat#4423 | |
Chemical compound, drug | Wnt-C59 | Cayman | Cat#16644 | |
Chemical compound, drug | Accutase | Sigma-Aldrich | Cat#SCR005 | |
Chemical compound, drug | Insulin | Gibco | Cat#12585014 | |
Chemical compound, drug | B-27 | Thermo Fisher | Cat#17504044 | |
Chemical compound, drug | Molecular Probes Fura-2 | Thermo Fisher | Cat#F1221 | |
Chemical compound, drug | Pluronic F-127 | Thermo Fisher | Cat#P6867 | |
Antibody | Anti-human-FN1 (Rabbit polyclonal) | Thermo Fisher | Cat#PA5-29578 | WB(1:1000) |
Antibody | Anti-rabbit IgG | Cell Signaling Technology | CST #7074 | WB(1:400) |
Chemical compound, drug | KAPA HiFi HS | KAPA | Cat#KK2502 | |
Commercial assay, kit | PrimeFlow RNA Assay Kit | Thermo Fisher | Cat#88-18005-210 | |
Chemical compound, drug | NEBNext High-Fidelity | New England Biolabs | Cat#M0541L | |
Chemical compound, drug | Gibson Assembly Master Mix | New England Biolabs | Cat#E2611L | |
Chemical compound, drug | mTeSR Plus | Stemcell Technologies | Cat#100–0276 | |
Chemical compound, drug | Matrigel | Corning | Cat#354277 | |
Commercial assay, kit | P3 Primary Cell 4D-Nucleofector X Kit | Lonza | Cat#V4XP-3024 | |
Commercial assay, kit | 10× genomics Chromium Single Cell 3′ Kit V3.1 | 10× Genomics | Cat#PN-1000147 | |
Commercial assay, kit | 10× CellPlex | 10× Genomics | Cat#PN-1000261 | |
Commercial assay, kit | 10× Target Hybridization Kit | 10× Genomics | Cat#PN-1000248 | |
Chemical compound, drug | RPMI 1640 | Thermo Fisher | Cat#11875093 | |
Chemical compound, drug | KnockOut Serum | Thermo Fisher | Cat#10828028 | |
Chemical compound, drug | HHBSS | Corning | Cat#21-023-CM | |
Strain, strain background (Endura) | Endura ElectroCompetent Cells | Lucigen | Cat#60242–2 | Electrocompetent Cells |
Strain, strain background (Escherichia coli) | Stellar Competent Cells | Clontech | Cat#636766 | |
Other | Single-cell RNA-seq Data | This paper | GEO: GSE190475 | Sequencing data located at GEO |
Cell line (Homo sapiens) | 293T cells | ATCC | CRL-3216 | Mycoplasma free; ATCC STR authenticated as CRL-3216 |
Cell line (Homo sapiens) | H9 cells | WiCell | WA09 | Mycoplasma free; ATCC STR authenticated as WAe009-A-18 |
Recombinant DNA reagent | Plasmid: pMD2.G | Addgene | RRID:Addgene_12259 | |
Recombinant DNA reagent | Plasmid: psPAX2 | Addgene | RRID:Addgene_12260 | |
Recombinant DNA reagent | Plasmid: lenti-dCas9-KRAB-Blast | Addgene | RRID:Addgene_89567 | |
Recombinant DNA reagent | Plasmid: CROPseq-Guide-puro | Addgene | RRID:Addgene_86708 | |
Sequence-based reagent | sgRNA oligos | This paper | sgRNA oligos | Supplementary file 1 |
Sequence-based reagent | qPCR primers | This paper | qPCR primers | Supplementary file 2 |
Software, algorithm | Star | PMID:23104886 | RRID:SCR_004463 | |
Software, algorithm | Picard | Broad Institute | RRID:SCR_006525 | |
Software, algorithm | FlowCal | PMID:27110723 | RRID:SCR_018140 | |
Software, algorithm | FeatureCounts | DOI:10.1093/bioinformatics/btt656 | RRID:SCR_012919 | |
Software, algorithm | 10× Genomics Cellranger | 10× Genomics | RRID:SCR_023221 | |
Software, algorithm | Scanpy | PMID:29409532 | RRID:SCR_018139 | |
Software, algorithm | IGV | Broad Institute | RRID:SCR_011793 | |
Other | Illumina NextSeq 500 instrument | Illumina | Next-generation sequencer | |
Other | Illumina NextSeq 2000 instrument | Illumina | Next-generation sequencer | |
Other | Illumina NovaSeq 6000 instrument | Illumina | Next-generation sequencer | |
Other | Agilent 2200 TapeStation instrument | Agilent | Automated electrophoresis instrument | |
Other | Qubit Fluorometric Quantitation instrument | Thermo Fisher | Flurometer | |
Other | EVOS FL Auto Imaging System | Thermo Fisher | Microscope |
List of target enhancers and sequences used in study.
Sequencing statistics.
Top 100 Louvain cluster defining genes for each dataset.
Differentially expressed genes through hypergeometric test.
Focus screen mid-CM and late-CM defining genes.
Differentially expressed genes between TBX5 enhancer 5 heterozygous KO cells and WT cells, in the late-CM state.