Assembly line biosynthesis creates numerous structurally diverse natural products using a common modular synthetic strategy. The collinearity between the architectures of modular polyketide synthases (PKS) and the structures of their polyketide products would seem to render these biosynthetic machineries excellent platforms for designer biosynthesis, yet reliable strategies to reprogram these assembly lines without diminishing their activities have not been identified. Here, as a best practice for PKS engineering, we demonstrate the reprogramming of the mediomycin PKS without significant loss of productivity. Using in vitro CRISPR/Cas9 gene editing followed by heterologous expression, we reconstruct an inaccessible drug lead of the fibrinogen receptor, tetrafibricin, at 82 ± 3 mg/L yield, retaining 26% productivity after five-step module editing using an evolution-supported cut site, downstream of the acyltransferase domain. A macrocyclic aminopolyol is also accessed through thioesterase swapping. These results pave the way toward the rational reprogramming of PKSs to access desired complex organic molecules. The collinearity between the architectures of modular polyketide synthases (PKS) and the structures of their polyketide products would suggest these biosynthetic machineries are excellent platforms for designer biosynthesis, yet reliable strategies to reprogram these assembly lines without diminishing their activities have not been identified. Here, the authors demonstrate the reprogramming of the mediomycin PKS without significant loss of productivity, and reconstruct an inaccessible drug lead of the fibrinogen receptor, tetrafibricin, at 82 mg/L yield.

Engineering proteins with desired functions remains challenging and usually requires multiple rounds of screening and selection. Here, we present Sequence Display, a platform that generates large-scale protein sequence–activity datasets in a single round. Sequence Display enables multiplexed assessment of individual variant activity within a single experiment, offering a robust approach to mapping detailed sequence–function relationships. We demonstrate the platform’s broad applicability by generating datasets for cytosine deaminase, uracil glycosylase inhibitor, aminoacyl-tRNA synthetase and a compact Cas9 nuclease. Integrating these datasets obtained from Sequence Display with pretrained protein language models, fine-grained, variant-specific activity landscapes can be constructed. We discovered several Cas9 variants with expanded protospacer-adjacent motif recognition and evolved aminoacyl-tRNA synthetase variants capable of recognizing different noncanonical amino acids. Together, this study establishes Sequence Display as a powerful tool for mapping protein activity landscapes and accelerating the discovery of optimized proteins for biological and medical applications. Sequence Display maps protein variant activities to a sequencing-based readout.

Type VII CRISPR-Cas system, evolutionarily associated with type III systems, utilizes a Cascade complex formed by Cas5 and catalytically inactive Cas7 copies for target RNA binding, but instead incorporates a specialized Cas14 ribonuclease for target cleavage. Here, we report a high-quality cryo-EM structure at the target engagement state with a shortened crRNA and elucidate how the recruited Cas14 captures the target RNA and undergoes target-mediated activation. The signature Cas14 is homologous to eukaryotic CPSF73 and prokaryotic RNase J, comprising two conserved subdomains, MβL and β-CASP. Different from canonical type III systems, 5′-end target RNA, rather than 3′-end, is bent into the positively charged binding channel formed by the two subdomains to access the conserved catalytic pocket on Cas14. Two special structural features, α1 helix from Cas7 and α10 helix from Cas14, promote the bent target RNA docking into the catalytic pocket of Cas14 nuclease in concert. A dual-functional loop, displaced by the entering target RNA, induces a closed-to-open transition between the two subdomains for nuclease activation. More importantly, the flipped dual-functional loop also maintains the stabilization of incoming target RNA. Altogether, our work provides a more comprehensive understanding of type VII system mechanism, laying a mechanistic foundation for RNA-targeting tool development.

Bacterial exposure to constant phage attack drives rapid diversification of antiphage defense systems, often through the exchange of modular defensive domains. Here, we leverage this modularity signature to identify new defense systems by systematically searching for operons encoding known defensive domains in non-canonical configurations. Using this approach, we identified 39 848 candidate defense operons in E. coli genomes. Annotation of the operons based on their shared defensive domains with known systems reveals that the operons represent variants of 82 defense families. Experimental testing of nine candidates validated six with anti-phage activity. These include DarTG and ietAS system variants that have acquired helicase modules, and a Gabija system in which a MazF-like protein replaces GajA, implying novel anti-phage mechanisms. We also identified a new clade of Pycsar that synergizes with type IV Thoeris to broaden phage protection. Our findings demonstrate that mining modular defensive domains provides a powerful strategy to predict and characterize new anti-phage systems, expanding the known repertoire of bacterial immunity.

The 5′-repeat fragments released during pre-crRNA maturation are critical yet understudied components of the CRISPR/Cas12a system. Here, we demonstrate that engineered 5′-repeat fragments can potently activate Cas12a cleavage, with efficiency strongly dependent on the length of the 3′-spacer. Strikingly, complete truncation of the 3′-spacer generates a “chiral-like crRNA” conformation that induces a delayed-switch mode of Cas12a activation, fundamentally distinct from conventional mature crRNA. Leveraging this characteristic, we develop the delayed cleavage feature-mediated one-pot sensing strategy that resolves the long-standing challenge of incompatibility between Cas12a-based cleavage reaction and nucleic acid amplification, achieving a 1000-fold improvement in sensitivity over that of the conventional mature crRNA-mediated one-pot method. Furthermore, we integrate a cleavage-based one-pot assay with a portable temperature-controlled fluorescence imaging device to create an on-site diagnostic platform for high-throughput screening. Our study further advances the understanding of the crRNA-guided mechanism and facilitates the expansion of its applications in genome editing and molecular diagnostics. In CRISPR/Cas12a systems, small RNA from pre-crRNA maturation is often overlooked. Here, authors show these fragments reconstitute with Cas12a to activate cleavage. They discover a crRNA activating Cas12a in a “delayed-switch” mode, resolving its incompatibility with nucleic acid amplification.

The 3D architecture and dynamics of the genome are crucial for regulation of genome stability, transcription and cellular function. CRISPR-based live imaging technologies have enabled real-time visualization of specific genomic loci and transcripts in living cells. These tools harness customized guide RNAs and nuclease-deactivated Cas effectors to achieve precise genomic targeting, and recent methodological advances provide the 3D spatiotemporal resolution required to decipher real-time chromatin communication. These methods are elucidating the biophysical properties of chromatin, linking dynamic enhancer–promoter interactions directly to transcription, and revealing the role of 3D genome dynamics in basic cellular processes and disease. Here, we summarize the development of CRISPR-based live-cell imaging techniques, highlight the complementary 3D microscopy and analysis methods compatible with these methods, and offer perspectives on their applications to uncover fundamental principles that govern genome dynamics and function. In this Review, Zhu et al. outline the various CRISPR-based tools recently built for dynamic DNA and RNA imaging, which have provided insights into various molecular mechanisms. The authors also discuss the important parameters to consider when using these tools, and how to approach the quantitative image analysis.

Similar to proteins, many RNAs fold into three-dimensional (3D) structures to perform biological functions. Here we present the trRosettaRNA server, a web-based platform for automated RNA 3D structure prediction using deep learning. The primary input is the nucleotide sequence of a target RNA, with the option to upload custom multiple sequence alignments and secondary structures. The server uses an end-to-end neural network for automated 3D structure prediction, followed by an energy optimization step to resolve structural violations. As an automated server, trRosettaRNA is distinguished by its state-of-the-art modeling accuracy, flexible input options and comprehensive visualization of prediction results. trRosettaRNA has been successfully applied in various contexts, including predicting structures for Rfam families lacking known 3D structures, where representative cases of high-confidence structure predictions were found to align well with subsequent experimental observations. Utilizing up to 5 central processing unit (CPU) cores in parallel on our computer cluster, the server takes a median time of about 1 h to predict structures for RNA sequences with about 200 nucleotides. The standalone package for trRosettaRNA offers distinct advantages such as enhanced data privacy for sensitive sequences, the ability to bypass server queues and integration into high-throughput automated pipelines. Importantly, the open-source nature of the package empowers researchers to directly modify the codebase for specialized research needs or to develop derivative tools by fine-tuning the underlying neural network. The web server and standalone package of trRosettaRNA are available at https://yanglab.qd.sdu.edu.cn/trRosettaRNA/ and https://github.com/YangLab-SDU/trRosettaRNA2 , respectively. The trRosettaRNA server is a web-based platform for automated and accurate RNA 3D structure prediction using deep learning. This protocol also describes how to use the standalone package locally, which is beneficial for large-scale applications.

Functional metagenomics has emerged as an effective tool for discovering novel enzymes directly from environmental samples, overcoming the limitations of traditional culture-based methods. In this study, we used a functional metagenomic approach on stool samples from Axis kuhlii, an endemic deer species from Indonesia, to identify active cellulases. We created an efficient workflow for expression of metagenomic sequences directly in Komagatella phaffii by combining metagenomic sequencing to investigate enzyme diversity, multiplex PCR to build a genes library, and rolling circle amplification (RCA) to streamline the cloning process, eliminating the need for intermediate Escherichia coli transformation and propagation steps. Furthermore, a semi-high-throughput screening method was used to evaluate multiple samples at once, allowing for the rapid identification of active enzymes. Using this approach, we discovered five endoglucanases and three β-glucosidases with confirmed enzyme activity. This study shows that functional metagenomics can bridge the gap between computational predictions and experimental validation, providing a reliable platform for enzyme discovery and characterization from complex environmental microbiomes. 

Transcription factors (TFs) efficiently locate their target DNA sequences by combining three-dimensional diffusion and one-dimensional sliding on nonspecific DNA. To balance rapid sliding with strong specific binding, TFs were proposed to switch between search and recognition conformations. For E. coli lac repressor (LacI), the folding of the hinge helices has been implicated in the conformational switch. Here, we tested how mutations in the hinge region impact the search speed and binding stability. Based on molecular dynamics simulations, we selected two LacI mutants favoring either search or recognition conformation. We measured the binding kinetics of the mutants both in vitro on DNA microarrays with 2479 different Lac operators and in vivo via single-molecule experiments. We identified a mutation that enhances the specificity but reduces binding strength globally, and another mutation that makes the operator binding stronger but also reduces the specificity. However, the altered specificity impacts the search time less than expected. Instead, the major effect was impaired dissociation in response to Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction for the strongly binding mutant. Together with earlier reports of affinity–inducibility trade-offs in LacI, our data support the model in which the trade-off is between binding stability and inducibility rather than between speed and binding stability.

Cell-free systems have great potential for nucleic acid assays, yet universal onsite diagnostics without preamplification are limited. Here, a universal cell-free diagnostic platform termed TRACKer (Target-Responsive non-preAmplification Cell-free diagnostic Kit) has been developed for detecting multiple respiratory viral RNAs in laboratory and field settings. TRACKer integrates three modules: ribozyme allostery module, riboregulator activation module, and output module. The ribozyme allostery module enables universal target recognition through strand displacement-mediated ribozyme conformational switching. The riboregulator activation module achieves preamplification-free detection via cascade expression of reporter proteins. The output module provides swappable reporter templates for luminescent quantification and lateral flow visualization in diverse scenarios. TRACKer is a robust system that enables rapid ( < 70 min) nucleic acid detection without preamplification and demonstrates attomolar sensitivity (1-10 aM) for six respiratory viruses. Overall, TRACKer presents a promising approach to nucleic acid detection, offering a low-cost and scalable solution with potential applications in point-of-care diagnostics and beyond. Cell-free systems hold potential for nucleic acid assays but are limited by the need for pre-amplification. Here the authors develop a cell-free diagnostic platform that detects viral RNA without pre-amplification demonstrating uses on six respiratory viruses in under 70 minutes with attomolar sensitivity.

Cell-type-specific promoters are used in gene therapy to restrict expression of the therapeutic payload. However, these promoters often have suboptimal strength, selectivity and size. Here, leveraging recent insights into the function of enhancers, we developed synthetic super-enhancers (SSEs) by assembling functionally validated enhancer fragments into multipart arrays. Focusing on the core SOX2-driven and SOX9-driven transcriptional regulatory network in glioblastoma stem cells (GSCs)1, we engineered SSEs with robust activity and high selectivity. Single-cell profiling, biochemical analyses and genome-binding data indicated that SSEs integrate neurodevelopmental and signalling-state transcription factors to trigger the formation of large multimeric complexes of transcription factors. Moreover, GSC-selective expression of a combination of cytotoxic (HSV-TK and ganciclovir) and immunomodulatory (IL-12) payloads, delivered using adeno-associated virus vectors, as a single treatment led to curative outcomes in a mouse model of aggressive glioblastoma. Notably, IL-12 induced an immunological memory that prevented tumor recurrence. The activity and selectivity of the adeno-associated virus and SSE were validated using primary human glioblastoma tissue and normal cortex samples. In summary, SSEs harness the unique core transcriptional programs that define the GSC phenotype and enable precision immune activation. This approach may have broader applications in other contexts when precise control of transgene expression in specific cell states is necessary. Synthetic super-enhancers enable specific delivery of anticancer payloads, achieving tumour elimination after a single dose in a mouse model of aggressive glioblastoma.

Acinetobacter baumannii natural isolates encode multiple copies of E. coli DNA polymerase V (pol VEc) umuDC homologs, some with as many as four nonidentical umuC and three nonidentical umuD genes encoding twelve possible pol VAb variants. Here, we show that six of the twelve pol VAb drive spontaneous and methyl methanesulfonate-induced mutations when expressed in E. coli Mutagenesis depends on co-expression with RecAAb. Five mutagenically active pol VAb combinations assemble in vitro as stable mutasome complexes that synthesize DNA, pol VAb Mut = UmuD′2CAb-RecAAb-ATP/ATPγS. One of the mutasomes requires an A. baumannii-encoded β/τ processivity clamp for activity in vitro. Translesion DNA synthesis (TLS) occurs at T^T cyclobutane dimers, with different variants exhibiting different nucleotide misincorporation specificities. As observed for E. coli pol V and R391 ICE-encoded Rum pol, a single amino acid substitution in RecA, RecAAb M196D, abolishes A. baumannii pol V-induced mutagenesis.