CRISPR/CAS9 ablation of individual miRNAs from a miRNA family reveals their individual efficacies for regulating cardiac differentiation.

ABSTRACT

Although it is well understood that genetic mutations, chromosomal abnormalities, and epigenetic miscues can cause congenital birth defects, many defects are still labeled idiopathic, meaning their origin is not yet understood. microRNAs are quickly entering the causal fray of developmental defects. miRNAs use a 7-8 base-pair seed sequence to target a corresponding sequence on one or multiple mRNAs resulting in rapid down-regulation of translation. miRNAs can also control protein 'amounts' in cells. As a result if miRNAs are over or under expressed during development protein homeostasis can be compromised resulting in defects in the development of organ systems. Here, we show that during differentiation of embryonic stem cells, individual miRNAs that reside in the miRNA17 family (composed of 14 miRNAs) do not share the same function even though they have the same seed sequence. The advent of CRISPR/CAS9 technology has not only yielded a true observation of individual miRNA function, it has also reconnected advanced molecular biology approaches to classical cell biology approaches such as gene rescue. We show that miRNA106a and to a lesser extent miR17 and 93 target the cardiac suppressor gene Fog2, which specifically suppress Gata-4 and Coup-TF2. However, when each miRNA is knocked out, we find that their targeting efficacies for Fog2 differ resulting in varying degrees of cardiac differentiation.