Precise base editing without unintended indels in human cells and mouse primary myoblasts.

Base editors are powerful tools for making precise single-nucleotide changes in the genome. However, they can lead to unintended insertions and deletions at the target sites, which is a significant limitation for clinical applications. In this study, we aimed to eliminate unwanted indels at the target sites caused by various evolved base editors. Accordingly, we applied dead Cas9 instead of nickase Cas9 in the base editors to induce accurate substitutions without indels. Additionally, we tested the use of chromatin-modulating peptides in the base editors to improve nucleotide conversion efficiency. We found that using both dead Cas9 and chromatin-modulating peptides in base editing improved the nucleotide substitution efficiency without unintended indel mutations at the desired target sites in human cell lines and mouse primary myoblasts. Furthermore, the proposed scheme had fewer off-target effects than conventional base editors at the DNA level. These results indicate that the suggested approach is promising for the development of more accurate and safer base editing techniques for use in clinical applications.

Connexin 43 is required for the maintenance of mitochondrial integrity in brown adipose tissue.

We investigated the role of connexin 43 (Cx43) in maintaining the integrity of mitochondria in brown adipose tissue (BAT). The functional effects of Cx43 were evaluated using inducible, adipocyte-specific Cx43 knockout in mice (Gja1 adipoq KO) and by overexpression and knockdown of Cx43 in cultured adipocytes. Mitochondrial morphology was evaluated by electron microscopy and mitochondrial function and autophagy were assessed by immunoblotting, immunohistochemistry, and qPCR. The metabolic effects of adipocyte-specific knockout of Cx43 were assessed during cold stress and following high fat diet feeding. Cx43 expression was higher in BAT compared to white adipose tissue. Treatment with the ß3-adrenergic receptor agonist CL316,243 increased Cx43 expression and mitochondrial localization. Gja1 adipoq KO mice reduced mitochondrial density and increased the presence of damaged mitochondria in BAT. Moreover, metabolic activation with CL316,243 further reduced mitochondrial integrity and upregulated autophagy in the BAT of Gja1 adipoq KO mice. Inhibition of Cx43 in cultured adipocytes increased the generation of reactive oxygen species and induction of autophagy during ß-adrenergic stimulation. Gja1 adipoq KO mice were cold intolerant, expended less energy in response to ß3-adrenergic receptor activation, and were more insulin resistant after a high-fat diet challenge. Collectively, our data demonstrate that Cx43 is required for maintaining the mitochondrial integrity and metabolic activity of BAT.