Targeted protein degradation in mammalian cells: A promising avenue toward future.

In the eukaryotic cellular milieu, proteins are continuously synthesized and degraded effectively via endogenous protein degradation machineries such as the ubiquitin-proteasome and lysosome pathways. By reengineering and repurposing these natural protein regulatory mechanisms, the targeted protein degradation (TPD) strategies are presenting biologists with powerful tools to manipulate the abundance of proteins of interest directly, precisely, and reversibly at the post-translational level. In recent years, TPD is gaining massive attention and is recognized as a paradigm shift both in basic research, application-oriented synthetic biology, and pioneering clinical work. In this review, we summarize the updated information, especially the engineering efforts and developmental route, of current state-of-the-art TPD technology such as Trim-Away, LYTACs, and AUTACs. Besides, the general design principle, benefits, problems, and opportunities to be addressed were further analyzed, with the aim of providing guidelines for exploration, discovery, and further application of novel TPD tools in the future.

Biosynthesis of pinene in purple non-sulfur photosynthetic bacteria.

BACKGROUND: Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored. RESULTS: Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 µg/L of pinene was yielded. Then, genes of 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 µg/L. CONCLUSIONS: In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application.

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.

Bioinformatic Identification of miR-622 Key Target Genes and Experimental Validation of the miR-622-RNF8 Axis in Breast Cancer.

Breast cancer is the leading cause of cancer-associated deaths among females. In recent decades, microRNAs (miRNAs), a type of short non-coding RNA that regulates gene expression at the post-transcription level, have been reported to participate in the regulation of many hub genes associated with tumorigenesis, tumor progression, and metastasis. However, the precise mechanism by which miRNAs regulate breast cancer metastasis remains poorly discussed, which limits the opportunity for the development of novel, effective therapeutic targets. Here, we aimed to determine the miR-622-related principal regulatory mechanism in cancer. First, we found that miR-622 was significantly related to a poor prognosis in various cancers. By utilizing an integrated miRNA prediction process, we identified 77 promising targets and constructed a protein-protein interaction network. Furthermore, enrichment analyses, including GO and KEGG pathway analyses, were performed to determine the potential function of miR-622, which revealed regulation networks and potential functions of miR-622. Then, we identified a key cluster comprised of six hub genes in the protein-protein interaction network. These genes were further chosen for pan-cancer expression, prognostic and predictive marker analyses based on the TCGA and GEO datasets to mine the potential clinical values of these hub genes. To further validate our bioinformatic results, the regulatory axis of miR-622 and RNF8, one of the hub genes recently reported to promote breast cancer cell EMT process and breast cancer metastasis, was selected as in vitro proof of concept. In vitro, we demonstrated the direct regulation of RNF8 by miR-622 and found that the predicted miR-622-RNF8 axis could regulate RNF8-induced epithelial-mesenchymal transition, cell migration, and cell viability. These results were further demonstrated with rescue experiments. We established a closed-loop miRNA-target-phenotype research model that integrated the bioinformatic analysis of the miRNA target genes and experimental validation of the identified key miRNA-target-phenotype axis. We not only identified the hub target genes of miR-622 in silico but also revealed the regulatory mechanism of miR-622 in breast cancer cell EMT process, viability, and migration in vitro for the first time.

Avenues Toward microRNA Detection In Vitro: A Review of Technical Advances and Challenges.

Over the decades, the biological role of microRNAs (miRNAs) in the post-transcriptional regulation of gene expression has been discovered in many cancer types, thus initiating the tremendous expectation of their application as biomarkers in the diagnosis, prognosis, and treatment of cancer. Hence, the development of efficient miRNA detection methods in vitro is in high demand. Extensive efforts have been made based on the intrinsic properties of miRNAs, such as low expression levels, high sequence homology, and short length, to develop novel in vitro miRNA detection methods with high accuracy, low cost, practicality, and multiplexity at point-of-care settings. In this review, we mainly summarized the newly developed in vitro miRNA detection methods classified by three key elements, including biological recognition elements, additional micro-/nano-materials and signal transduction/readout elements, their current challenges and further applications are also discussed.

Assembly of TALE-based DNA scaffold for the enhancement of exogenous multi-enzymatic pathway.

Synthetic scaffold systems, which exhibit enzyme clustering effect, have been considered as an important parallel approach for metabolic flux control and pathway enhancement. Here, we described an improved DNA-based scaffold system for synthetic tri-enzymatic pathway in Escherichia coli. With plasmid DNA serving as scaffold and exogenous enzymes fused with rationally designed transcription activator-like effectors (TALEs), our approach successfully clustered three TALE-fused enzymes and significantly increased the production of a mevalonate-producing tri-enzymatic pathway with the optimized scaffold structure and plasmid copy number. These results further suggested the scalability and robustness of the TALE-based scaffold system, and we can assume that it can be used on numerous multi-enzyme metabolic pathways due to its programmable features.

Spatial organization of enzymes to enhance synthetic pathways in microbial chassis: a systematic review.

For years, microbes have been widely applied as chassis in the construction of synthetic metabolic pathways. However, the lack of in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. In recent years, multiple methods have been applied to the construction of small metabolic clusters by spatial organization of heterologous metabolic enzymes. These methods mainly focused on using engineered molecules to bring the enzymes into close proximity via different interaction mechanisms among proteins and nucleotides and have been applied in various heterologous pathways with different degrees of success while facing numerous challenges. In this paper, we mainly reviewed some of those notable advances in designing and creating approaches to achieve spatial organization using different intermolecular interactions. Current challenges and future aspects in the further application of such approaches are also discussed in this paper.

Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9.

MicroRNAs have been reported as related to multiple diseases and have potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost, and low sensitivity as of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on rolling circle amplification, CRISPR-Cas9, and split-horseradish peroxidase techniques. It is able to detect trace amount of miRNAs from samples with mere single-base specificity. Moreover, we demonstrated that such scheme can effectively detect target miRNAs in clinical serum samples and significantly distinguish patients of non-small cell lung cancer from healthy volunteers by detecting the previously reported biomarker: circulating let-7a. As the first to use CRISPR-Cas9 in miRNA detection, this method is a promising approach capable of being applied in screening, diagnosing, and prognosticating of multiple diseases.

Spatial organization of heterologous metabolic system in vivo based on TALE.

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.