The functional coherence between microRNAs (miRNAs) and their host genes remains enigmatic. As host genes and its intronic miRNAs are transcribed as cistronic units, their functional coherence could be beneficial for the organism. However, it is not clear how broad the coherence takes place in the organisms. In this study, we focused on the functions of a miRNA miR-140 and its host gene Wwp2, both of which are reported to be important for the craniofacial development in mouse (Miyaki et al. Gene Dev. 2010, Zou et al. Nat. Cell Biol. 2011). Using CRISPR/Cas9 system, we generated single and double knockout mice for miR-140 and Wwp2 to explore the possible coherence in their functions. Unexpectedly, we found that the miRNA, but not the protein coding gene, is involved in the craniofacial development. Further analysis with compound heterozygous mice suggested that the gene-trap cassette used in the original report of Wwp2 knockout mouse (Zou et al. Nat. Cell Biol. 2011) unintendedly downregulated the expression of miR-140, which lies downstream of the insertion site, and this caused the craniofacial phenotype. Our results dissected the role of a microRNA and its host gene by CRISPR/Cas9 system, and raised a caution for the phenotypic evaluation of knockout mice generated by gene-trap or similar strategy.

miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3' UTR of a luciferase gene. Using this reporter library system, we conducted a screening for targets of miR-34a, a tumor-suppressor miRNA. We identified both previously characterized and previously uncharacterized targets. miR-34a overexpression in MDA-MB-231 breast cancer cells repressed the expression of these previously unrecognized targets. Among these targets, GFRA3 is crucial for MDA-MB-231 cell growth, and its expression correlated with the overall survival of patients with breast cancer. Furthermore, GFRA3 was found to be directly regulated by miR-34a via its coding region. These data show that this system is useful for elucidating miRNA functions and networks.