Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.
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The diverse functions of thyroid hormones are thought to be mediated by two nuclear receptors, T3Rα1 and T3Rβ, encoded by the genes T3Rα and T3Rβ respectively. The T3Rα gene also produces a non‐ligand‐binding protein T3Rα2. The in vivo functions of these receptors are still unclear. We describe here the homozygous inactivation of the T3Rα gene which abrogates the production of both T3Rα1 and T3Rα2 isoforms and that leads to death in mice within 5 weeks after birth. After 2 weeks of life, the homozygous mice become progressively hypothyroidic and exhibit a growth arrest. Small intestine and bones showed a strongly delayed maturation. In contrast to the negative regulatory function of the T3Rβ gene on thyroid hormone production, our data show that the T3Rα gene products are involved in up‐regulation of thyroid hormone production at weaning time. Thus, thyroid hormone production might be balanced through a positive T3Rα and a negative T3Rβ pathway. The abnormal phenotypes observed on the homozygous mutant mice strongly suggest that the T3Rα gene is essential for the transformation of a mother‐dependent pup to an ‘adult’ mouse. These data define crucial in vivo functions for thyroid hormones through a T3Rα pathway during post‐natal development.
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