Thyroid hormone (T3) affects adult metabolism and postembryonic development in vertebrates. T3 functions mainly via binding to its receptors (TRs) to regulate gene expression. There are 2 TR genes, TRα and TRβ, with TRα more ubiquitously expressed. During development, TRα expression appears earlier than T3 synthesis and secretion into the plasma. This and the ability of TRs to regulate gene expression both in the presence and absence of T3 have indicated a role for unliganded TR during vertebrate development. On the other hand, it has been difficult to study the role of unliganded TR during development in mammals because of the difficulty to manipulate the uterus-enclosed, late-stage embryos. Here we use amphibian development as a model to address this question. We have designed transcriptional activator-like effector nucleases (TALENs) to mutate the TRα gene in Xenopus tropicalis. We show that knockdown of TRα enhances tadpole growth in premetamorphic tadpoles, in part because of increased growth hormone gene expression. More importantly, the knockdown also accelerates animal development, with the knockdown animals initiating metamorphosis at a younger age and with a smaller body size. On the other hand, such tadpoles are resistant to exogenous T3 treatment and have delayed natural metamorphosis. Thus, our studies not only have directly demonstrated a critical role of endogenous TRα in mediating the metamorphic effect of T3 but also revealed novel functions of unliganded TRα during postembryonic development, that is, regulating both tadpole growth rate and the timing of metamorphosis.
Gene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9’s homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a “plug and play” module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.
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