[Generation of tnnt2a knock-out zebrafish via CRISPR/Cas9 and phenotypic analysis]

Cardiac troponin T (cTnT) serves as a structural protein of myocardial fiber, and participates in heart excitation-contraction coupling process. Here, we generated tnnt2a (cTnT-coding gene) deletion mutant zebrafish via CRISPR/Cas9 technique, and performed phenotypic analysis of the identified tnnt2a mutants. We observed that there was no significant difference between heterozygous mutant and wild type zebrafish, and the homozygous mutants displayed significant malformations in heart, including cardiac arrest, atrium and ventricle enlargement, pericardium effusion, and the individuals usually died before 7 day post fertilization (dpf). We further analyzed the expression alternations of heart sarcomere genes (tnnt2a, actc1a, tpm4a, myl7, vmhc) at transcriptional level in tnnt2a^-/-(Δ2) zebrafish by performing real time RT-PCR, and found that the RNA expression level of tnnt2a in tnnt2a^-/- zebrafish decreased constantly at each time point of developmental stages, and actc1a, tpm4a, myl7 and vmhc all showed higher expressions at early developmental stages and lower expressions at late developmental stages, in comparison with those of wild type zebrafish. Lastly, electron microscopy on cardiac tissues suggested that there were significant changes of the thick or thin filament structures in tnnt2a^-/-(Δ2) zebrafish, which was further confirmed by F-actin and Tpm4 immunofluorescence staining. The tnnt2a^-/- zebrafish generated by CRISPR/Cas9 bears the most common symptoms of patients with dilated cardiomyopathy, and therefore can be used as a tool to study TNNT2-deficiency related cardiomyopathy.