Fig. 3

(A) A publicly available dataset was used to populate a list of candidate genes enriched in the embryonic zebrafish heart. ~14% of the genes (orange dots) have reported cardiac phenotypes in ZFIN suggesting enrichment of genes important in heart development. (B) Filtering to remove genes with known mutant phenotypes yields 188 poorly-characterized genes potentially important for cardiovascular development in zebrafish. (C) gRNA sequences with fewer off-targets were prioritized. (D) MIC-Drop screen of the 188 candidate genes and subsequent phenotyping shows no significant differences in viability between uninjected and droplet-injected embryos by 3 dpf N = (Left panel: Uninjected-1801, Injected-2502; Right panel: Uninjected-1571, Injected-2013). Embryos with gross morphological defects at 3 dpf (~15%) were removed and the barcodes of those with cardiac defects were sequenced. Droplets targeting npas4l were spiked-in at 2% proportion as positive control. (E) Barcode sequencing of embryos displaying any cardiac phenotype (e.g. looping defect, chamber dysmorphogenesis, valve defect, arrhythmia, etc.) yields “hit” candidates. Heat map shows the observed frequency of each barcode. As positive controls, barcodes for tnnt2a, nkx2.5, and npas4l are enriched in the set of embryos exhibiting any visible cardiac phenotype. Genes with barcode frequency of ≥ 4 (Binomial probability ≤ 0.05) or with consistent cardiac phenotypes were considered for secondary validation. (F) Secondary validation by direct RNP injection corroborates screening results and identifies a dozen novel genes, the loss of which results in cardiac phenotypes in at least 20% of F0 embryos. N = (WT-238; actb2/actb1-124; alad-137; atp6v1c1-135; clec19a-182; cox8a-130; ddah2-126; fam171a1-259; gpr78a-186; gse1-266; gstm.3-199; ppan-304; rbm4.3-153; sf3b4-307; sntb1-107; tpte-209; wasf3a/b-130; vmhc-107; gata4-106; cx36.7-147; nkx2.5-186; tnnt2a-124; npas4l-103).