Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana.

Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.

Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy.

Gene targeting (GT) is a promising tool for precise manipulation of genome sequences, however, GT in seed plants remains a challenging task. The simple and direct way to improve the efficiency of GT via homology-directed repair (HDR) is to increase the frequency of double-strand breaks (DSBs) at target sites in plants. Here we report an all-in-one approach of GT in Arabidopsis by combining a transcriptional and a translational enhancer for the Cas expression. We find that facilitating the expression of Cas9 and Cas12a variant by using enhancers can improve DSB and subsequent knock-in efficiency in the Arabidopsis genome. These results indicate that simply increasing Cas protein expression at specific timings - egg cells and early embryos - can improve the establishment of heritable GTs. This simple approach allows for routine genome engineering in plants.

Corydalis saxicola Bunting Total Alkaloids ameliorate diet-induced non-alcoholic steatohepatitis by regulating hepatic PI3K/Akt and TLR4/NF-κB pathways in mice.

Corydalis saxicola Bunting (Yanhuanglian), distributed in Southwest China, is mainly used for treatment of hepatitis, oral mucosal erosion, conjunctivitis, dysentery, acute abdominal pain and hemorrhoids in the folk. Corydalis saxicola Bunting Total Alkaloids (CSBTA) are the active ingredients extracted from the root of C. saxicola bunting. Non-alcoholic steatohepatitis (NASH) is the hinge between steatosis and cirrhosis in the spectrum of Non-alcoholic fatty liver disease (NAFLD), which has become one of the most common chronic liver diseases in the world. CSBTA can reduce tumors and brain diseases through anti-inflammatory and antioxidant pathways. Our study was designed to clarify the effects of CSBTA on the HFHC (High fat and high carbohydrate drinking) diet induced mice. In our research, A HFHC diet induced NASH mice model was applied to investigate the effects of CSBTA in vivo and obeticholic acid (OA) was set as positive control. Moreover, the underlying mechanisms were explored by palmitic acid (PA) and lipopolysaccharide (LPS) stimulated HepG2 cells in vitro. The in vivo study illustrated that CSBTA could alleviate mice away from the onset of NASH, and reduce intrahepatocellular lipid accumulation and hepatocyte inflammation under high fat condition. Further in vitro analysis confirmed that CSBTA attenuated inflammation and hepatic lipid accumulation by improving hepatic PI3K/Akt and suppressing hepatic TLR4/NF-κB pathways. In summary, this study demonstrated that CSBTA might be a promising compound for the treatment of NAFLD.