Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations.

Highlights d Statin treatment among 13,981 patients with COVID-19 was retrospectively studied d Statin use in this cohort was associated with a lower risk of all-cause mortality d Adding an ACE inhibitor or an ARB did not affect statinassociated outcome in the cohort d The benefit of statins among this cohort may be due to immunomodulatory benefits

Background and Aims: COVID-19 is a dominant pulmonary disease, with multisystem involvement, depending upon co morbidities. Its pro le in patients with pre-existing chronic liver disease (CLD) is largely unknown. We studied the liver injury patterns of SARS-Cov-2 in CLD patients, with or without cirrhosis. Methods: Data was collected from 13 Asian countries on patients with CLD, known or newly diagnosed, with con rmed COVID-19. Result: Altogether, 228 patients [185 CLD without cirrhosis and 43 with cirrhosis] were enrolled, with comorbidities in nearly 80%. Metabolism associated fatty liver disease (113, 61%) and viral etiology (26, 60%) were common. In CLD without cirrhosis, diabetes [57.7% vs 39.7%, OR=2.1(1.1-3.7), p=0.01] and in cirrhotics, obesity, [64.3% vs. 17.2%, OR=8.1(1.9-38.8), p=0.002) predisposed more to liver injury than those without these. Forty three percent of CLD without cirrhosis presented as acute liver injury and 20% cirrhotics presented with either acute-on-chronic liver failure [5(11.6%)] or acute decompensation [4(9%)]. Liver related complications increased (p<0.05) with stage of liver disease; a Child-Turcotte Pugh score of 9 or more at presentation predicted high mortality [AUROC-0.94, HR=19.2(95CI 2.3-163.3), p<0.001, sensitivity 85.7% and speci city 94.4%). In decompensated cirrhotics, the liver injury was progressive in 57% patients, with 43% mortality. Rising bilirubin and AST/ALT ratio predicted mortality among cirrhosis. Conclusions: SARS-Cov-2 infection causes signi cant liver injury in CLD patients, decompensating one fth of cirrhosis, and worsening the clinical status of the already decompensated. The CLD patients with diabetes and obesity are more vulnerable and should be closely monitored.

With rapid lifestyle transitions, the increasing burden of nonalcoholic fatty liver disease (NAFLD) in China has emerged as a major public health issue. To obtain a comprehensive overview of the status of NAFLD over the past decade, we evaluated the epidemiology, risk factors, complications, and management of NAFLD in China through a systematic review and meta‐analysis. Five English literature databases and three Chinese databases were searched for relevant topics from 2008 to 2018. A total of 392 studies with a population of 2,054,554 were included. National prevalence of NAFLD was 29.2%, with a heavier disease burden among the middle‐aged, males, those in northwest China and Taiwan, regions with a gross domestic product per capita greater than 100,000 yuan, and Uygur and Hui ethnic groups. Currently, original studies on natural history and complications of NAFLD in China are scarce. Several studies revealed that NAFLD is positively correlated with the incidence of extrahepatic tumors, diabetes, cardiovascular disease and metabolic syndrome. The Chinese population may have a higher hereditary risk of NAFLD due to more frequent nonsynonymous mutations in genes regulating lipid metabolism. Ultrasonography is the primary imaging tool in the detection of NAFLD in China. Serum tests and risk stratification algorithms for staging NAFLD remain under investigation. Specific pharmaceutical treatments for NAFLD are still undergoing clinical trials. It is noteworthy that the Chinese are underrepresented compared with their proportion of the NAFLD population in such trials. Conclusion: China experienced an unexpected rapid increase in the burden of NAFLD over a short period. Rising awareness and urgent actions need to be taken in order to control the NAFLD pandemic in China.

With dramatic changes in lifestyles over the last 20 years, nonalcoholic fatty liver disease (NaFlD) has become the most prevalent liver disorder in China but has not received sufficient attention. NaFlD-related advanced liver disease and its mortality along with its overall disease burden are expected to increase substantially. there is thus an imperative need to clarify the epidemiological features of NaFlD to guide a holistic approach to management. We summarize eight epidemiological features of NaFlD in China over the past two decades using systematic review and meta-analysis methodology. our data reveal a rapid growth in the NaFlD population, especially among younger individuals. Importantly, there is a strong ethnic difference in body mass index (BMI) and genetic risk of NaFlD compared with the US population. the etiology of advanced liver disease and its complications (e.g., hepatocellular carcinoma) has been altered because of a Westernized lifestyle and the implementation of effective vaccination strategies against viral hepatitis. Regional epidemiological patterns of NaFlD indicate that economics, environment, and lifestyle are critical factors in disease progression. the analysis also indicates that a large number of patients remain undiagnosed and untreated because of the inadequacy of diagnostic tools and the absence of effective pharmacologic therapies. given the burden of NaFlD, future policy and research efforts need to address knowledge gaps to mitigate the risk burden.

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

BackgroundDynamic N6-methyladenosine (m6A) modification was previously identified as a ubiquitous post-transcriptional regulation that affected mRNA homeostasis. However, the m6A-related epitranscriptomic alterations and functions remain elusive in human cancer. Here we aim to identify the profile and outcome of m6A-methylation in hepatocellular carcinoma (HCC).ResultsUsing liquid chromatography-tandem mass spectrometry and m6A-immunoprecipitation in combination with high-throughput sequencing, we determined the m6A-mRNA levels in human HCC. Human HCC exhibited a characteristic gain of m6A modification in tandem with an increase of mRNA expression, owing to YTH domain family 2 (YTHDF2) reduction. The latter predicted poor classification and prognosis of HCC patients, and highly correlated with HCC m6A landscape. YTHDF2 silenced in human HCC cells or ablated in mouse hepatocytes provoked inflammation, vascular reconstruction and metastatic progression. Mechanistically, YTHDF2 processed the decay of m6A-containing interleukin 11 (IL11) and serpin family E member 2 (SERPINE2) mRNAs, which were responsible for the inflammation-mediated malignancy and disruption of vascular normalization. Reciprocally, YTHDF2 transcription succumbed to hypoxia-inducible factor-2α (HIF-2α). Administration of a HIF-2α antagonist (PT2385) restored YTHDF2-programed epigenetic machinery and repressed liver cancer.ConclusionOur results have characterized the m6A-mRNA landscape in human HCC and revealed YTHDF2 as a molecular ‘rheostat’ in epitranscriptome and cancer progression.