As an alternative and complementary approach to Cas9-based genome editing, Cas12a has not been widely used in mammalian cells largely due to its strict requirement for the TTTV protospacer adjacent motif (PAM) sequence. Here, we report that Mb3Cas12a (Moraxella bovoculi AAX11_00205) can efficiently edit the mouse genome based on the TTV PAM sequence with minimal numbers of large on-target deletions or insertions. When TTTV PAM sequencetargeting CRISPR (cr)RNAs of 23 nt spacers are used, >70% of the founders obtained are edited. Moreover, the use of Mb3Cas12a tagged to monomeric streptavidin (mSA) in conjunction with biotinylated DNA donor template leads to high knock-in efficiency in two-cell mouse embryos, with 40% of founders obtained containing the desired knock-in sequences.

Livin, a member of the inhibitor of apoptosis protein (IAP) family, is expressed at a high level in lung adenocarcinoma and influences the progression of cancer, and its response to chemotherapy and radiotherapy. Aberrant microRNA (miRNA) expression has also been associated with cancer initiation and development. However, the clinical significance of Livin and its relationship with miRNAs in lung adenocarcinoma are still unclear. In the present study, the expression level of Livin in 90 pairs of lung adenocarcinoma and their adjacent tissues were detected by immunohistochemistry staining. Spearman correlation and Kaplan-Meier, univariate and multivariate analyses were applied to evaluate the correlation between the expression of Livin and clinical characteristics. With the integration of bioinformatics analysis and dual-luciferase reporter gene assays, we identified the miRNA that can target Livin mRNA. The functional effects of miRNA-mediated Livin knockdown were assessed by Cell Counting Kit-8 (CCK-8) and apoptosis assays, and cell cycle analysis. The present study revealed that Livin was upregulated in lung adenocarcinoma tissues and may be associated with the poor prognosis in lung adenocarcinoma patients. The overexpression of Livin is partly caused by the downregulation of miR-198. Further exploration revealed that miRNA-198-mediated silencing of Livin significantly inhibited cell growth and enhanced apoptosis of A549 cells, accompanied by marked upregulation of caspase-3. Finally, we observed that the miR-198 overexpression and Livin neutralization had similar effects on improving cisplatin chemosensitivity in A549 cells. Overall, these findings suggest that Livin has the potential to become a biomarker for predicting the prognosis of lung adenocarcinoma and may provide a promising strategy for assisting chemotherapy of lung adenocarcinoma through the miR-198/Livin/caspase-3 regulatory network.

MYCT1 is an important gene known to regulate cell viability and apoptosis of laryngeal cancer cells. However, the underlying molecular mechanism remains unclear. Here, we show that MAX enhances the expression of miR‐181a by directly binding to its promoter, whereas miR‐181a targets NPM1 and suppresses its expression in laryngeal cancer cells. MYCT1 and miR‐181a decrease cell viability and colony formation through enhanced apoptosis, whereas NPM1 displays opposite effects in laryngeal cancer cells. Their opposing functions are further supported by the findings (a) that miR‐181a is down‐regulated, while NPM1 is up‐regulated in laryngeal cancer, and (b) that either inhibition of miR‐181a or overexpression of NPM1 can revert the pro‐apoptotic effects of MYCT1 on laryngeal cancer cells through extracellular and intracellular apoptotic pathways. Our data suggest that MYCT1 may synergistically interact with MAX as a co‐transcription factor or a component of MAX transcriptional complex, to transcriptionally regulate the expression of miR‐181a, which, in turn, decreases NPM1 expression at post‐transcriptional levels, leading to enhanced apoptosis in laryngeal cancer cells. These factors may serve as potential targets for early diagnosis and treatment of laryngeal cancer.

Perinatal exposure to smoking has been associated with childhood asthma, one of the most common pediatric conditions affecting millions of children globally. Of great interest, this disease phenotype appears heritable as it can persist across multiple generations even in the absence of persistent exposure to smoking in subsequent generations. Although the molecular mechanisms underlying childhood asthma induced by perinatal exposure to smoking or nicotine remain elusive, an epigenetic mechanism has been proposed, which is supported by the data from our earlier analyses on germline DNA methylation (5mC) and histone marks (H3 and H4 acetylation). To further investigate the potential epigenetic inheritance of childhood asthma induced by perinatal nicotine exposure, we profiled both large and small RNAs in the sperm of F1 male rats. Our data revealed that perinatal exposure to nicotine leads to alterations in the profiles of sperm-borne RNAs, including mRNAs and small RNAs, and that rosiglitazone, a PPARγ agonist, can attenuate the effect of nicotine and reverse the sperm-borne RNA profiles of F1 male rats to close to placebo control levels.