A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs.

Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed 'Self-targeting sgRNA Library Screen' (SLS) for assaying the activity of Cas9 nucleases in bacteria using random target/sgRNA libraries of self-targeting sgRNAs. Exploiting more than a million different sequences, we demonstrate the use of the method with the SpCas9-HF1 variant to analyse its activity and reveal motifs that influence its target-selectivity. We have also developed an algorithm for predicting the activity of SpCas9-HF1 with an accuracy matching those of existing tools. SLS is a facile alternative to the much more expensive and laborious approaches used currently and has the capability of delivering sufficient amount of data for most of the orthologs and variants of SpCas9.

Sex influenced association of directly measured insulin sensitivity and serum transaminase levels: Why alanine aminotransferase only predicts cardiovascular risk in men?

BACKGROUND: Non alcoholic fatty liver disease (NAFLD) is an independent cardiovascular (CV) risk factor which is closely associated with insulin resistance measured by both direct or indirect methods. Gender specific findings in the relationship between alanine aminotransferase (ALT) and CV disease, the prevalence of NAFLD and type 2 diabetes (T2DM) have been published recently. The aim of the present study was to explore the gender aspects of the association between insulin sensitivity, liver markers and other metabolic biomarkers in order to elucidate the background behind the sex influenced difference in both NAFLD, T2DM and their association with CV risk. PATIENTS AND METHODS: 158 female (47 normal and 111 impaired glucose intolerant) and 148 male (74 normal and 74 impaired glucose tolerant) subjects were included (mean age: 46.5 ± 8.31 vs. 41.6 ± 11.3, average Hba1c < 6.1 %, i.e. prediabetic population, drug naive at the time of the study). Subjects underwent a hyperinsulinemic normoglycemic clamp to determine muscle glucose uptake (M3), besides liver function tests and other fasting metabolic and anthropometric parameters were determined. RESULTS: Significant bivariate correlations were found between clamp measured M3 and all three liver enzymes (ALT, aspartate aminotransferase and gamma-glutamyl transferase) in both sexes. When data were adjusted for possible metabolic confounding factors correlations ceased in the male population but stayed significant in the female group. Feature selection analysis showed that ALT is an important attribute for M3 in the female but not in male group (mean Z: 3.85 vs. 0.107). Multiple regression analysis confirmed that BMI (p < 0.0001) and ALT (p = 0.00991) significantly and independently predicted clamp measured muscle glucose uptake in women (R(2) = 0.5259), while in men serum fasting insulin (p = 0.0210) and leptin levels (p = 0.0294) but none of the liver enzymes were confirmed as significant independent predictors of M3 (R(2) = 0.4989). CONCLUSION: There is a gender specific association between insulin sensitivity, metabolic risk factors and liver transaminase levels. This might explain the sex difference in the predictive role of ALT elevation for CV disease. Moreover, ALT may be used as a simple diagnostic tool to identify insulin resistant subjects only in the female population according to our results.