Nature Protocols最新文献

CRISPR screens have revolutionized the study of diverse biological processes, particularly in cancer research. Both pooled and arrayed CRISPR screens have facilitated the identification of essential genes for cell survival and proliferation, drivers of drug resistance and synthetic lethal interactions. However, applying loss-of-function CRISPR screening to non-proliferative states remains challenging, largely because of slower editing and the poor sensitivity of identifying guide RNAs that 'drop out' in a population of non-dividing cells. Here, we present a detailed protocol to accomplish this, using an inducible Cas9 system that offers precise temporal control over Cas9 expression. This inducible system allows gene editing to occur only after the non-proliferative state is fully established. We describe the complete procedure for generating an inducible Cas9-expressing model and for measuring editing efficiency by using flow cytometry. In addition, we discuss how to optimize key parameters for performing successful CRISPR screens in various non-proliferative states. We describe a detailed workflow for performing a screen in senescent cells to identify senolytic targets. This protocol is accessible to researchers with experience in molecular biology techniques and can be completed in 8-12 weeks, from the generation of an inducible Cas9 cell line clone to the analysis of a CRISPR screen for hit identification. These techniques can be applied by researchers across different fields, including stem cell differentiation, immune cell development, aging and cancer research.

Transcranial electrical stimulation (tES) has gained substantial momentum as a research and therapeutic tool; however, it suffers from challenges related to reproducibility and quality assessment due to the absence of standardized reporting practices. Here we aim to develop a comprehensive and consensus-based checklist for conducting and reporting tES studies to enhance the quality of research and reports. In this Consensus Statement, we used a Delphi approach conducted across three rounds and involving 38 experts to identify crucial elements required to report in tES studies. This consensus-driven approach included the evaluation of the interquartile deviation (>1.00), the percentage of positive responses (above 60%) and mean importance ratings (<3), hence ensuring the creation of a robust and well-balanced checklist. These metrics were utilized to assess both the consensus reached and importance ratings for each item. Consensus was reached, leading to the retention of 66 out of the initial 70 items. These items were categorized into five groups: participants (12 items), stimulation device (9 items), electrodes (12 items), current (12 items) and procedure (25 items). We then distilled a shorter version of the checklist, which includes the 26 items deemed essential. The Report Approval for Transcranial Electrical Stimulation (RATES) checklist is relevant to those carrying out and assessing tES studies, as it provides a structured framework for researchers to consider and report. For reviewers, it can serve as a tool to assess completeness, comprehensiveness and transparency of reports. In addition, the RATES checklist aims to promote a deeper understanding of tES and facilitates comparisons between studies within the field. Overall, the RATES checklist provides a shared reference point that may improve research quality, foster harmonization in reporting and, ultimately, enhance the interpretability and reproducibility of findings in both research and clinical contexts.

Complex carbohydrates are essential to life processes, but it is challenging to isolate these molecules from natural sources in high homogeneity. Therefore, complex-glycan synthesis becomes critical to improving our understanding of their important functions. Due to their complexity, synthesis is still difficult for nonexperts. One of the key challenges is to search for general solutions for highly 1,2-cis-selective glycosylation, which will directly assemble 1,2-cis-2-aminoglycosides that are incorporated in numerous biologically important complex glycans and glycoconjugates. Here we describe an iron-catalyzed, chemical glycosylation method for rapid assembly of 1,2-cis-aminoglycosidic linkages. The iron catalyst is commercially available, and the bench-stable supporting ligand and amination reagents are easily prepared from abundant, readily available starting materials. This catalytic, exclusively 1,2-cis-selective glycosylation is effective for a broad range of glycosyl donors and acceptors, and it can be operated in a continuous fashion and scaled up to the multigram scale. The reactivity of this glycosylation is tunable for both electron-rich and electron-deficient substrates by modulating amination reagents. The glycosylation proceeds through a unique mechanism in which the iron catalyst activates a glycosyl acceptor and an oxidant when it facilitates the cooperative atom transfer of both moieties to a glycosyl donor in an exclusively cis-selective manner. This glycosylation protocol takes several hours to operate. It complements the existing 1,2-cis-selective glycosylation methods and effectively addresses the challenge of achieving both generality and high stereoselectivity in the 1,2-cis-selective aminoglycosylation.

Understanding the redox properties and catalytic behavior of proteins is critical for harnessing their functions in biocatalysis and to promote efficient bio-inspired catalysts design. Enzymatic X-ray absorption spectroelectrochemistry (XA-SEC) combines the insights of X-ray absorption spectroscopy with the precision of electrochemical methods to elucidate enzymes' redox properties and catalytic behavior. Here we describe how to perform enzymatic XA-SEC experiments. The procedure begins with the preparation of the carbon-based working electrode to enhance enzyme immobilization. We exemplify with the efficient immobilization of bilirubin oxidase from Myrothecium verrucaria on the electrode surface, utilizing nanomaterials to enhance biomaterial loading and electron-transfer at the enzyme-electrode interface. Next, we guide researchers through setting up a standard three-electrode electrochemical cell, ensuring proper electrical connections and electrolyte preparation. Our Protocol details the Cu K-edge X-ray absorption spectroscopy measurement procedure at the synchrotron light sources, with in situ electrochemical control. Real-time redox processes are monitored through direct electron transfer analysis, providing valuable thermodynamic and kinetic information. It is important to determine the stability and activity of the analyzed protein under X-ray beam exposure; our approach typically results in stable electrochemical and spectroscopic signals for long experimental runs, showcasing the enzyme's robust performance and efficient protein immobilization. The method's ability to correlate XA-SEC data with direct electron transfer and substrate-biding analysis provides a powerful tool for advancing our understanding of enzymatic electrocatalysis and opens new avenues for developing sustainable bioelectrochemical technologies.

Bioinspired in-sensor computing devices can process information at sensory terminals by leveraging physical principles, thereby reducing latency and energy consumption during computation while simultaneously enhancing the efficiency of data processing and real-time analysis. Optoelectronic devices exhibit in-sensor computing functions, such as feature enhancement and data compression, by tuning the defect states of the semiconductor channels and thereby modulating the photoresponsivity and time constants of the sensors. These functionalities are critically dependent on precise fabrication and testing protocols. Here we present a detailed procedure for fabricating and characterizing in-sensor computing devices based on nanoscale semiconductor thin films. We explain how to test such optoelectronic devices, including the testing of visual adaptation and motion perception responses. When using semiconductor materials obtained from commercial suppliers, this procedure is time efficient and results in highly reproducible device performance. Nevertheless, all device fabrication and testing steps are generalizable and can be extended to other semiconductor thin films grown using different methods. The procedure is intended for researchers experienced in cleanroom operations and microfabrication techniques and can be completed in ~14 d. The use of bioinspired optoelectronic devices enables the development of a framework for advancing in-sensor computing technologies.

Structural variants (SVs) contribute significantly to genomic diversity and disease predisposition as well as development in diverse species. However, their accurate characterization has remained a challenge because of their complexity and size. With the rise of third-generation sequencing technology, analytical strategies to map SVs have been revisited, and software such as NanoVar, a free and open-source package designed for efficient and reliable SV detection in long-read sequencing data, has facilitated their studies. NanoVar has been shown to work effectively in various published genomic studies, including research on genetic disorders, population genomics and genome analysis of non-model organisms. In this article, we describe in detail all the steps of the NanoVar protocol and its interplay with other platforms for SV calling in whole-genome long-read sequencing data such that researchers with minimal experience with command-line interfaces can easily carry out the protocol. It also provides exhaustive instructions for diverse study designs, including single-sample analyses, cohort studies and genome instability analyses. Finally, the protocol covers SV visualization, filtering and annotation details. Overall, users can identify and analyze SVs in a typical human dataset with a conventional computational setup in ~2-5 h after read mapping.

Controlled gene expression programs have a crucial role in shaping cellular functions and activities. At the core of this process lies the RNA life cycle, ensuring protein products are synthesized in the right place at the right time. Here we detail an integrated protocol for imaging-based highly multiplexed in situ profiling of spatial transcriptome using antibody-based protein comapping (STARmap PLUS), spatial translatome mapping (RIBOmap) and spatiotemporal transcriptome mapping (TEMPOmap). These methods selectively convert targeted RNAs, ribosome-bound mRNAs or metabolically labeled RNAs to DNA amplicons with gene-unique barcodes, which are read out through in situ sequencing under a confocal microscope. Compared with other methods, they provide the analytical capacity to track the spatial and temporal dynamics of thousands of RNA species in intact cells and tissues. Our protocol can be readily performed in laboratories experienced in working with RNA and equipped with confocal microscopy instruments. The wet lab experiments in preparing the amplicon library take 2-3 d, followed by variable sequencing times depending on the sample size and target gene number. The spatially resolved single-cell profiles enable downstream analysis, including cell type classification, cell cycle identification and determination of RNA life cycle kinetic parameters through computational analysis guided by the established tutorials. This spatial omics toolkit will help users to better understand spatial and temporal RNA dynamics in heterogeneous cells and tissues.

Photoluminescence imaging is valuable for elucidating biological processes and diagnosing diseases, but its tissue penetration is limited. We developed an imaging technique that utilizes ultrasound to activate the piezoelectric effect of a molecular probe, transforming ultrasound energy into chemical energy. The chemical energy is then converted into light emission through the chemiluminescence effect, improving penetration depth and overcoming traditional photoluminescence imaging constraints. Here we describe how to build two kinds of ultrasound-induced luminescence imaging systems. We introduce a procedure for the synthesis of trianthracene derivative (TD) nanoparticles with ultrasound-induced luminescence properties. The TDs are converted into water-soluble nanoparticles by a simple nanoprecipitation method. Utilizing the constructed ultrasound-induced luminescence imaging systems, TD nanoparticles can be stimulated to exhibit a luminescence spectrum peaking between 625 and 650 nm. Under optimized ultrasound excitation time and excitation power density parameters, the imaging quality and tissue penetration depth are effectively enhanced. Notably, our procedure enables the detection of both subcutaneous tumor models and challenging deep-tissue orthotopic gliomas. This ultrasound-mediated approach represents an important advancement over conventional photoluminescence imaging methods, enabling high-fidelity in vivo tumor imaging with superior signal quality. Establishment of the ultrasound-induced luminescence imaging systems requires ~2 h, the synthesis of TD molecules requires ~4 d, nanoparticle preparation requires ~1 d, ex vivo characterization requires ~1 d, investigation of the ultrasound-induced luminescence of TD nanoparticles requires ~3 d and ultrasound-induced luminescence imaging takes ~1 d. These steps can be performed by operators trained in chemical synthesis, nanomaterial synthesis standards and qualified in relevant animal experiments.

CRISPR-Cas9 technology has transformed the study of gene function, enabling the systematic investigation of host-virus interactions. However, most CRISPR-based screens in the context of viral infections rely on cell survival as a readout, which limits their sensitivity and biases results toward early infection stages. To address these challenges, we developed the virus-encoded CRISPR-based direct readout system (VECOS), a virus-centric approach in which human cytomegalovirus is engineered to express single-guide RNA (sgRNA) libraries directly from its genome. This system allows sgRNA abundance, embedded in the viral genome, to serve as a direct and quantitative readout of gene-perturbation effects on viral propagation. By tracking sgRNA levels at distinct stages of the viral infection cycle, VECOS enables a detailed, multidimensional analysis of virus-host interactions. Here we present a modular detailed Protocol for (1) constructing and reconstituting complex sgRNA libraries in double-stranded DNA viruses using bacterial artificial chromosomes, (2) performing multipassage screens to investigate perturbation effects on various stages of viral infection and (3) analyzing the multipassage and multistage sgRNA abundance measurements utilizing a comprehensive framework for data analysis. Successful implementation of this full Protocol takes 14-22 weeks and requires proficiency in molecular biology, as well as basic familiarity with Unix-based computing and programming in R for data processing. This Protocol offers researchers a robust tool for uncovering the molecular mechanisms that drive viral propagation and host-virus interactions.