Fig. 2

An orthogonal transposase approach is required to efficiently generate cardiomyocyte-specific crispants (A) Representation of the tnnt2a locus in the zebrafish genome, indicating the exons targeted by the selected gRNAs (asterisks). (B–E) Confocal projections of representative 96 h post fertilization (hpf) embryonic hearts from wild-type (B and C) or cardiodeleter+ animals (D and E) from the indicated experimental groups, immunostained to detect cardiomyocyte nuclei (white) and Tnnt2a (cyan). The percentage of Tnnt2a-negative (Tnnt2a–) cardiomyocytes is summarized in the bottom left corner (average ± SD, n = 4, 7, 6, and 8 embryos). (F and J) Confocal projections of 96 hpf embryonic hearts from animals carrying the cardiodeleter transgene and injected with a Tol2- (F; n = 8) or Tol1- (J; n = 7) based guide shuttle encoding gRNAs targeting tnnt2a, immunostained to detect Tnnt2a (cyan). Yellow and white arrowheads indicate Tnnt2a+ and Tnnt2a– guide shuttle+ cardiomyocytes (magenta), respectively. Single confocal planes are shown in F′ and J′. (G–I) Representative ventricular sections from ∼30 dpf cardiodeleter+ animals, uninjected (G) or injected with the indicated combinations of guide shuttles and mRNA (H and I). White arrows indicate cardiomyocyte nuclei. Yellow arrows and cyan arrowheads indicate mKate+cardiodeleter+ and mKate+cardiodeleter– cardiomyocytes, respectively. Cartoons (right) summarize the observed patterns of excision and integration of the cardiodeleter and guide shuttles. Chr, chromosome. Scale bars, 20 μm.