Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species.

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Inhibition kinetics of acetosyringone on xylanase in hydrolysis of hemicellulose.

Many phenolic compounds, derived from lignin during the pretreatment of lignocellulosic biomass, could obviously inhibit the activity of cellulolytic and hemicellulolytic enzymes. Acetosyringone (AS) is one of the phenolic compounds produced from lignin degradation. In this study, we investigated the inhibitory effects of AS on xylanase activity through kinetic experiments. The results showed that AS could obviously inhibit the activity of xylanase in a reversible and noncompetitive binding manner (up to 50% activity loss). Inhibitory kinetics and constants of xylanase on AS were conducted by the HCH-1 model (ß = 0.0090 ± 0.0009 mM-1). Furthermore, intrinsic and 8-anilino-1-naphthalenesulfonic (ANS)-binding fluorescence results showed that the tertiary structure of AS-mediated xylanase was altered. These findings provide new insights into the role of AS in xylanase activity. Our results also suggest that AS was an inhibitor of xylanase and targeting AS was a potential strategy to increase xylose production.

Knockdown of creatine kinase B inhibits ovarian cancer progression by decreasing glycolysis.

Creatine kinase plays a key role in the energy homeostasis of vertebrate cells. Creatine kinase B (CKB), a cytosolic isoform of creatine kinase, shows upregulated expression in a variety of cancers. In this research, we confirmed that some ovarian cancer tissues had elevated CKB expression at the protein level. The functions of CKB in ovarian cancer progression were investigated in the ovarian cancer cell line Skov3, which has a high CKB expression. It was found that CKB knockdown inhibited Skov3 cell proliferation and induced apoptosis under hypoxia or hypoglycemia conditions. CKB depletion also sensitized Skov3 to chemotherapeutic agents. Furthermore, the CKB knockdown reduced glucose consumption and lactate production, and increased ROS production and oxygen consumption. This suggested that CKB knockdown decreased cytosolic glycolysis and resulted in a tumor suppressive metabolic state in Skov3 cells. Consequently, we found that the knockdown of CKB induced G2 arrest in cell cycle by elevating p21 expression and affected the PI3K/Akt and AMPK pathways. These findings provide new insights in the role of CKB in cancer cell survival and tumor progression. Our results also suggest that CKB depletion/inhibition in combination with chemotherapeutic agents might have synergistic effects in ovarian cancer therapy.