Comparison and optimization of different CRISPR/Cas9 donor-adapting systems for gene editing.

Gene knock-in in mammalian cells usually uses homology-directed repair (HDR) mechanism to integrate exogenous DNA template into the target genome site. However, HDR efficiency is often low, and the co-localization of exogenous DNA template and target genome site is one of the key limiting factors. To improve the efficiency of HDR mediated by CRISPR/Cas9 system, our team and previous studies fused different adaptor proteins with SpCas9 protein and expressed them. By using their characteristics of binding to specific DNA sequences, many different CRISPR/SpCas9 donor adapter gene editing systems were constructed. In this study, we used them to knock-in eGFP gene at the 3'-end of the terminal exon of GAPDH and ACTB genes in HEK293T cells to facilitate a comparison and optimization of these systems. We utilized an optimized donor DNA template design method, validated the knock-in accuracy via PCR and Sanger sequencing, and assessed the efficiency using flow cytometry. The results showed that the fusion of yGal4BD, hGal4BD, hLacI, hTHAP11 as well as N57 and other adaptor proteins with the C-terminus of SpCas9 protein had no significant effect on its activity. At the GAPDH site, the donor adapter systems of SpCas9 fused with yGal4BD, hGal4BD, hLacI and hTHAP11 significantly improved the knock-in efficiency. At the ACTB site, SpCas9 fused with yGal4BD and hGal4BD significantly improved the knock-in efficiency. Furthermore, increasing the number of BS in the donor DNA template was beneficial to enhance the knock-in efficiency mediated by SpCas9-hTHAP11 system. In conclusion, this study compares and optimizes multiple CRISPR/Cas9 donor adapter gene editing systems, providing valuable insights for future gene editing applications.

Diagnostic value of novel hepatic fibrosis markers in assessing cirrhosis in patients with chronic hepatitis C / 中华肝脏病杂志

Objective: To investigate the efficacy of chitinase-3-like protein 1 (CHI3L1) and Golgi protein 73 (GP73) in the diagnosis of cirrhosis and the dynamic changes of CHI3L1 and GP73 after HCV clearance in patients with chronic hepatitis C (CHC) treated with direct-acting antiviral drugs (DAAs). The comparison of continuous variables of normal distribution were statistically analyzed by ANOVA and t-test. The comparison of continuous variables of non-normal distribution were statistically analyzed by rank sum test. The categorical variables were statistically analyzed by Fisher's exact test and χ(2) test. Correlation analysis was performed using Spearman correlation analysis. Methods: Data of 105 patients with CHC diagnosed from January 2017 to December 2019 were collected. The receiver operating characteristic curve (ROC curve) was plotted to study the efficacy of serum CHI3L1 and GP73 for the diagnosis of cirrhosis. Friedman test was used to compare CHI3L1 and GP73 change characteristics. Results: The areas under the ROC curve for CHI3L1 and GP73 in the diagnosis of cirrhosis at baseline were 0.939 and 0.839, respectively. Serum levels of CHI3L1 and GP73 in the DAAs group decreased significantly at the end of treatment compared with baseline [123.79 (60.25, 178.80) ng/ml vs. 118.20 (47.68, 151.36) ng/ml, P = 0.001; 105.73 (85.05, 130.69) ng/ml vs. 95.52 (69.52, 118.97) ng/ml, P = 0.001]. Serum CHI3L1 and GP73 in the pegylated interferon combined with ribavirin (PR) group were significantly lower at the end of 24 weeks of treatment than the baseline [89.15 (39.15, 149.74) ng/ml vs. 69.98 (20.52, 71.96) ng/ml, P < 0.05; 85.07 (60.07, 121) ng/ml vs. 54.17 (29.17, 78.65) ng/ml, P < 0.05]. Conclusion: CHI3L1 and GP73 are sensitive serological markers that can be used to monitor the fibrosis prognosis in CHC patients during treatment and after obtaining a sustained virological response. Serum CHI3L1 and GP73 levels in the DAAs group decreased earlier than those in the PR group, and the serum CHI3L1 levels in the untreated group increased compared with the baseline at about two years of follow-up.

Mechanism of Prunellae Spica Extracts on Lipid Metabolism in ZDF Rats Based on AMPK/ACC Signalling Pathway / 中国实验方剂学杂志

Objective: To observe the effect of Prunellae Spica extracts (PS) on the lipid metabolism in Zuker Diabetes Fatty (ZDF) rats based on AMP-activated protein kinase/acetyl CoA carboxylase (AMPK/ACC) signaling pathway.Method: The 32 male ZDF (fa/fa) type 2 diabetic rats were randomly divided into model group,metformin group (180 mg·kg-1·d-1),and low and high-dose PS groups (12.25,24.5 mg·kg-1·d-1),with 8 in each group.8 male Zuker Lean (ZL) rats were selected as normal group.Body weight and fasting blood glucose were monitored at the 0th,4th and 8th weeks after administration.After 8 weeks,abdominal aorta blood was collected,serum was frozen at-20℃ by centrifugation,liver tissue was frozen at-80℃,fixed with 4% paraformaldehyde and embedded in paraffin.Serum triglyceride (TG),cholesterol (CHO),low density lipoprotein cholesterol (LDL-C) and free fatty acid (FFA) levels were measured by radioimmunoassay.Fat droplets in hepatocytes were measured by oil red O staining.Gene expressions of AMP-activated protein kinase-alpha 2(AMPKα2),Acetyl CoA carboxylase (ACC) in liver were detected by real-time polymerase chain reaction (Real-time PCR).Protein expressions of p-AMPKα were observed by immuno-histochemical (IHC) method.Result: Compared with the normal group,the T2DM model group showed significant increases in serum levels of TG,CHO,LDL-C,FFA and lipid droplets in hepatocytes.AMPKα2 mRNA expression was decreased,while ACC1 and ACC2 mRNA expressions were increased significantly.p-AMPKα protein expression in liver was decreased significantly (PPα2,down-regulation in mRNA expressions of ACC1 and ACC2,and up-regulation in protein expression of p-AMPKα(PPConclusion:PS can effectively improve liver lipid metabolism in ZDF rats.Its mechanism may be related to the regulation of AMPK/ACC signaling pathway in liver.