Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity.

BACKGROUND: The CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a). RESULTS: We created new AsCas12a variant termed "AsCas12a-Plus" with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity. CONCLUSIONS: Our discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Potential value and chemical characterization of gut microbiota derived nitrogen containing metabolites in feces from Periplaneta americana (L.) at different growth stages.

The American cockroach, Periplaneta americana (L.), is able to highly survive in various complicated environments around the globe, and often considered as a pest. In contrast, billions of P. americana have been massively reared in China and extensively used as a medicinal insect, due to its function for preventing and treating ulceration and heart failure. Considering the possibility that microbiota-derived metabolites could be an effective source to identify promising candidate drugs, we attempted to establish a rapid method for simultaneous determination of gut microbiota metabolites from medicinal insects. In this study, network pharmacology approach and ultra-performance liquid chromatography (UPLC) technique were employed to reveal the potential pharmacological activity and dynamics variation of nitrogen-containing metabolites (NCMs) originated from the gut microbiota of breeding P. americana at different growth stages. A metabolites-targets-diseases network showed that NCMs are likely to treat diseases such as ulceration and cancer. The analysis of NCMs' content with the growth pattern of P. americana indicated that the content of NCMs declined with P. americana aging. Both principal component analysis and orthogonal partial least squares discriminant analysis suggested that 8-hydroxy-2-quinolinecarboxylic acid and 8-hydroxy-3,4-dihydro-2(1H)-quinolinone are the potential differential metabolic markers for discriminating between nymphs and adults of P. americana. Moreover, the developed UPLC method showed an excellent linearity (R2 > 0.999), repeatability (RSD < 2.6%), intra- and inter-day precisions (RSD < 2.2%), and recovery (95.5%-99.0%). Collectively, the study provides a valuable strategy for analyzing gut microbiota metabolites from insects and demonstrates the prospects for discovering novel drug candidates from the feces of P. americana.

Combining miR-23b exposure with mesenchymal stem cell transplantation enhances therapeutic effects on EAE.

Bone marrow mesenchymal stem cells (BMSCs) have the immuno-modulatory capacity to ameliorate autoimmune diseases, such as multiple schlerosis (MS), systemic lupus erythematosus and rheumatoid arthritis. However, BMSC-mediated immunosuppression can be challenging to achieve. The efficacy of BMSC transplantation may be augmented by an adjuvant therapy. Here, we demonstrated that treatment of mice with experimental autoimmune encephalomyelitis (EAE), a model of MS, with BMSCs over-expressing microRNA (miR)-23b provided better synergistic and longer-term therapeutic effects than treatment with traditional BMSCs. Over-expression of miR-23b enhanced the ability of BMSCs to inhibit differentiation of Th17 cells and reduced IL-17 secretion. Compared to traditional BMSCs, the miR-23b over-expressing BMSCs (miR23b-BMSCs) exhibited enhanced secretion of tumor growth factor beta 1 (TGF-ß1), a cytokine that promotes the differentiation of regulatory T (Treg) cells. Pathologically, miR23b-BMSC transplantation delayed EAE progression, apparently by reducing the Th17/Treg cell ratio and inhibiting inflammatory cell infiltration across the blood-brain barrier, and thus slowing spinal cord demyelination. These results may lead to better utility of BMSCs as a treatment for autoimmune diseases.

Neural-specific distribution of transmembrane protein TMEM240 and formation of TMEM240-Body.

Mutation in TMEM240 is suggested to cause SCA21, but the specific mechanism has not been clarified. The subcellular localization, specific biological function, and corresponding mechanism of action of TMEM240 have also not been delineated. In this study, the mRNA and protein expression of TMEM240 were assessed using qPCR and western blotting, respectively. Live cell imaging was used to establish the sub-cellular location of TMEM240, and electron microscopy was used to determine the morphology and distribution of TMEM240 in the cell. TMEM240 was specifically expressed in the neurons. Exogenous TMEM240 formed a multilayered cell structure, which we refer to as TMEM240-Body (T240-Body). T240-Body was separated and purified by centrifugation and filtration. An anchor protein His-tagged-GFP-BP on Ni-NTA agarose was used to pull down T240-GFP binding proteins. Both the N-terminal and the C-terminal of TMEM240 were confirmed to be inside the T240-Body. Co-localization experiments suggested that peroxisomes might contribute to T240-Body formation, and the two transmembrane regions of TMEM240 appear to be essential for formation of the T240-Body. Emerin protein contributed to formation of T240-Body when combined with TMEM240. Overall, this study provides new insights into TMEM240, which inform future research to further our understanding of its biological function.

Differentiating prostate cancer from benign prostatic hyperplasia using PSAD based on machine learning: Single-center retrospective study in China.

The incidence of prostate cancer increases annually. Prostate cancer is an underreported and emerging problem in China. We conducted a cross-sectional study of 392 eligible patients from 710 men with prostate cancer or benign prostatic hyperplasia between 2000 and 2003. For total prostate-specific antigen, age, three diameters of prostate, prostate volume and prostate-specific antigen density seven indices, analysis of variance and t test were used to analyze the difference between the groups. A decision tree with pruning was established using the prostate-specific antigen density, age and transversal diameter of the prostate to screen the patient with prostate cancer. According to the established decision tree model, prostate-specific antigen density was the most important factor affecting the occurrence of prostate cancer. In elderly people over the age of 83 years, the transverse diameter of prostate cancer was smaller than that of benign prostatic hyperplasia, with prostate-specific antigen density less than . No additional index was introduced, and the detection rate of prostate cancer was 86.6 %.The specificity was enhanced to 78.1%.