Fig. 3

Cardiodeleter-induced crispants carry biallelic mutations in ∼90% of labeled cardiomyocytes (A) Representation of the location of the gRNAs targeting tnnt2a, cmlc2, and amhc. Tol1-guide shuttles encoding three guides for each gene were injected into wild-type or cardiodeleter+ embryos at the one-cell stage. Embryos exhibiting mKate+ clones in the heart, indicative of guide shuttle integration, were selected at 4 dpf and fixed for immunostaining. (B and C) Confocal projections of representative 96 h post fertilization embryonic hearts from wild-type (B) or cardiodeleter+ animals (C) injected with guide shuttles targeting the indicated genes, immunostained with specific antibodies to recognize the corresponding protein (cyan). Cyan arrowheads indicate guide shuttle (mKate)+ cardiomyocytes with biallelic mutations, leading to the complete loss of the corresponding protein. Yellow arrows indicate mKate+ cardiomyocytes maintaining protein expression. (D) Quantification of the efficiency of biallelic mutations in wild-type or cardiodeleter embryos injected with the indicated guide shuttles. Individual values plotted; black line: average. p values: two-tailed unpaired t test. AT, atrium; chr, chromosome; V, ventricle. Scale bars, 20 μm.