Fig. 5

A guide shuttle encoding multiple gRNAs targeting amhc is required for highly efficient biallelic mutagenesis (A–C) Confocal projections of representative 96 h post fertilization embryonic hearts from cardiodeleter+ animals injected with guide shuttles carrying three (A), two (B), or one (C) gRNAs targeting amhc. (D) Quantification of the efficiency of biallelic mutations in wild-type and cardiodeleter embryos injected with the indicated guide shuttles. Individual values plotted; black line: average. p values: one-way ANOVA, followed by Tukey’s multiple comparisons test. (E–G) Histological sections of hearts from adult cardiodeleter animals injected with the indicated guide shuttle, immunostained with antibodies to identify mKate (guide shuttle, magenta) and Amhc (cyan). Amhc signal is shown in (E′–G′). Cyan arrowheads indicate mKate (guide shuttle)+ cardiomyocytes with biallelic mutations that lead to complete depletion of the Amhc protein. Orange arrows indicate guide shuttle+ cardiomyocytes that maintain detectable levels of Amhc expression. (H) Efficiency of biallelic mutations in wild-type and cardiodeleter adult animals injected with the indicated guide shuttles. Individual values plotted; black line: average. p values: one-way ANOVA, followed by Tukey’s multiple comparisons test. (I) Distribution of guide shuttle+ cardiomyocyte population according to Amhc protein levels in wild-type and cardiodeleter adult animals injected with the indicated guide shuttles. at, atrium; v, ventricle. Scale bars, 20 μm (A–C) and 100 μm (E–G).