Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in Cupriavidus necator.

Cupriavidus necator H16 is a "Knallgas" bacterium with the ability to utilize various carbon sources and has been employed as a versatile microbial cell factory to produce a wide range of value-added compounds. However, limited genome engineering, especially gene regulation methods, has constrained its full potential as a microbial production platform. The advent of CRISPR/Cas9 technology has shown promise in addressing this limitation. Here, we developed an optimized CRISPR interference (CRISPRi) system for gene repression in C. necator by expressing a codon-optimized deactivated Cas9 (dCas9) and appropriate single guide RNAs (sgRNAs). CRISPRi was proven to be a programmable and controllable tool and could successfully repress both exogenous and endogenous genes. As a case study, we decreased the accumulation of polyhydroxyalkanoate (PHB) via CRISPRi and rewired the carbon fluxes to the synthesis of lycopene. Additionally, by disturbing the expression of DNA mismatch repair gene mutS with CRISPRi, we established CRISPRi-Mutator for genome evolution, rapidly generating mutant strains with enhanced hydrogen peroxide tolerance and robustness in microbial electrosynthesis (MES) system. Our work provides an efficient CRISPRi toolkit for advanced genetic manipulation and optimization of C. necator cell factories for diverse biotechnology applications.

Effect of sevoflurane on CD4+CD25+FOXP3+ regulatory T cells in patients with gastric cancer undergoing radical surgery.

In this study, the proportion of CD4+CD25+FOXP3+ regulatory T (Treg) cells in CD4+ T cells in the peripheral blood of gastric cancer patients before anesthesia induction (T1), after surgery (T2) and the first day after surgery (T3) was studied to explore the effect of sevoflurane and propofol anesthesia on the prognosis of gastric cancer patients. Forty patients with advanced gastric cancer were recruited and randomly divided into the sevoflurane group (S group) and the propofol group (T group). Flow cytometry was used to detect the proportion of CD4+CD25+FOXP3+ Treg cells in CD4+ T cells in the peripheral blood of patients with T1, T2 and T3, respectively. Compared with stage ⅡB, the proportion of CD4+CD25+FOXP3+ Treg cells in T1, T2 and T3 of stage ⅢA and stage ⅢB patients was increased. Compared with the T group, the expression of CD4+CD25+FOXP3+ Treg cells in the peripheral blood of T2 and T3 in the S group was decreased. The results showed that the expression of CD4+CD25+FOXP3+ Treg cells might be related to the TNM stage of gastric cancer and sevoflurane could alleviate the inhibition of postoperative immune function more than propofol. Sevoflurane effectively reduced the expression level of CD4+CD25+FOXP3+ Treg cells in peripheral blood of T2 and T3 of patients with gastric cancer, providing the theoretical basis for the selection of surgical anesthetics for patients with gastric cancer.

Comparative Analysis of Sample Extraction and Library Construction for Shotgun Metagenomics.

Human fecal specimens, serve as important materials, are widely used in the field of microbiome research, in which inconsistent results have been a pressing issue. The possible attribute factors have been proposed including the specimen status after preservation, extracted DNA quality, library preparation protocol, and sample DNA input. In this study, quality comparisons for shotgun metagenomics sequencing were performed between 2 DNA extraction methods for fresh and freeze-thaw samples, 2 library preparation protocols, and various sample inputs. The results indicate that Mag-Bind® Universal Metagenomics Kit (OM) outperformed DNeasy PowerSoil Kit (QP) with a higher DNA quantity. Controlling on library preparation protocol, OM detected on-average more genes than QP. For library construction comparison by controlling on the same DNA sample, KAPA Hyper Prep Kit (KH) outperformed the TruePrep DNA Library Prep Kit V2 (TP) with the higher number of detected genes number and Shannon index. No significant differences were found in taxonomy between 2 library preparation protocols using the fresh, freeze-thaw and mock community samples. No significant difference was observed between 250 and 50 ng DNA inputs for library preparation on both fresh and freeze-thaw samples. Through the preliminary study, a combined protocol is recommended for performing metagenomics studies, by using OM method plus KH protocol as well as suitable DNA quantity on either fresh or freeze-thaw samples. Our findings provide clues for potential variations from various DNA extraction methods, library protocols, and sample DNA inputs, which are critical for consistent and comprehensive profiling of the human gut microbiome.