Molecular engineering articles within Nature Communications

There are few robust circuit architectures for sequential gene perturbations. Here, the authors use a modular recombinase-based design that sequentially edits loci, synchronizes cells, and deletes itself.

CRISPR-based genetic elements can copy themselves across host genomes. Here the authors introduce CopyCatcher, a gene-drive related system for detecting and quantifying somatic gene conversion events.

Manipulating animal physiology can be difficult because of the complexity of multicellular systems. Here the authors use engineered bacteria to modulate Caenorhabditis elegans gene expression through genetic circuit controlled RNAi.

Transient transfections are routinely used in basic and synthetic biology studies to unravel pathway regulation and to probe and characterise circuit designs. As each experiment has a component of intrinsic variability, reporter gene expression is usually normalized with co-delivered genes that act as transfection controls. Recent reports in mammalian cells highlight how resource competition for gene expression leads to biases in data interpretation, with a direct impact on co-transfection experiments. Here we define the connection between resource competition and transient transfection experiments and discuss possible alternatives. Our aim is to raise awareness within the community and stimulate discussion to include such considerations in future experimental designs, for the development of better transfection controls.

Current aptamer discovery approaches are unable to probe the complete space of possible sequences. Here, the authors use machine learning to facilitate the development of DNA aptamers with improved binding affinities, and truncate them without significantly compromising binding affinity.

Base editors can induce transcriptome-wide RNA off-target editing independent of gRNA. Here, the authors engineer ABEmax variants with minimized RNA off-target activities.

Intrinsically disordered FG-Nups line the Nuclear Pore Complex (NPC) lumen and form a selective barrier where transport of most proteins is inhibited, whereas specific transporter proteins are able to pass. Here, the authors reconstitute the selective behaviour of the NPC by introducing a rationally designed artificial FG-Nup that demonstrates that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create selective NPCs.

Eukaryotic genetic switches are more complex than prokaryotic ones, complicating their design. Here the authors present a workflow for parallel screening, selection and evolution of yeast genetic switches.

Here, the authors present DisCo (Disassembly of Condensates), a method that allows the fast, inducible, and specific disruption of tagged condensates in mammalian cells. DisCo uses chemical dimerizers to induce the recruitment of a ligand into condensates leading to condensate disassembly.

Synthetic biology circuits are finding application in a wide range of computational devices, such as contaminant detection. Here, the authors design 2D paper circuits in which the spatial orientation of the cellular components specifies function.

Existing in vivo mutagenesis tools are limited by low mutation diversity and mutation rates. Here the authors present TRIDENT for targeted, continual and inducible diversification of genes of interest using deaminases fused to T7 RNA polymerase.

RaPID (Random non-standard Peptides Integrated Discovery) enables discovery of small macrocyclic peptides binding desired targets. Here, the authors propose lasso-grafting: the RaPID-derived peptides are implanted onto diverse proteins and maintain both the binding properties of the cyclic peptide and the host protein function.

NHEJ alleles and Cas9 remnants after a gene drive introduction are scientific and public concerns. Here, the authors use split drives with recoded rescue elements to target essential genes and minimize the appearance of NHEJ alleles while also leaving no trace of Cas9.

Biosensors are key to engineered biological systems. Here the authors demonstrate rapid de novo in vitro evolution of RNA biosensors of small molecules in a fully automated system.

Many diseases are caused by single-nucleotide polymorphisms. Here, the authors present CRISPR base editors that use the base excision machinery for single-base transversions.

Current developments in synthetic biology are not immediately applicable outside of the controlled laboratory environment. In this Perspective, the authors outline the advances and challenges the field faces in operating in resource limited and off-the-grid scenarios.

DNA has the potential to store vast amounts of data but it is subject to physical decay. In this Perspective, the authors propose that the stability of DNA should be a key consideration in how it is used for data storage.

Therapeutic application of RNA viruses requires tight control over viral activity. Here the authors design a regulatory switch that enables control over activity with clinically approved HIV protease inhibitors.

Advances in synthetic biology and genome engineering raise awareness of potential misuse. Here, the authors present PlasmidHawk, a sequence alignment based method for lab-of-origin prediction.

Direct cloning of biosynthetic gene clusters is difficult due to their size and repetitive nature. Here, the authors present CAPTURE, which uses Cas12a and in vivo Cre-lox recombination for efficient cloning of gene clusters up to 113 kb, and demonstrate how this method can be used for large-scale discovery of novel natural products.

Targeting a gene with two loxP sites is both time and labour intensive. Here the authors present Cre-Controlled CRISPR allowing conditional mutagenesis of a gene of interest and simultaneously labelling the putative mutant cells fluorescently.

Loss-of-function experiments are used in metabolic engineering to understand and optimise metabolism. Here, the authors apply auxin inducible protein degradation to test different metabolic engineering strategies for improved terpenoid production in yeast.

Coiled-coil protein origami is a strategy for the de novo design of polypeptide nanostructures based on coiled-coil dimer forming peptides, where a single chain protein folds into a polyhedral cage. Here, the authors design a single-chain triangular bipyramid and also demonstrate that the bipyramid can be self-assembled as a heterodimeric complex, comprising pre-defined subunits.

Histone phosphorylation is a ubiquitous post-translational modification. Here the authors present a programmable chromatin kinase, dCas9-dMSK1, that enables controlled histone phosphorylation and specific gene activation.

Getting synthetic biology circuit-based sensors into field applications is still a challenge. Here the authors combine a circuit sensor with a glucose meter for small analyte and nucleic acid detection.

Many next-generation antibody therapeutics have enhanced potency but the risk of adverse events. Here the authors develop a conditionally activated, single-module CAR.

Azoles are five-membered heterocycles found in peptidic natural products and synthetic peptiodomimetics. Here the authors demonstrate a posttranslational chemical modification method for in vitro ribosomal synthesis of peptides with exotic azole groups at specific positions.

Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in diverse cellular activities. Here the authors show a synthetic BRET-based transgene expression system that allows pulsatile and quantitative activation of gene expression both in live cells and in vivo.

Synthetic biologists often co-opt heterologous parts to affect new functions in living cells, yet such an approach has rarely been extended to structural components of the ribosome. Here, the authors describe generalizable methods to express ribosomes from divergent microbes in E. coli and maximize their function.

One of the key limitations of CRISPR-Cas-based genome editing techniques is the PAM dependency. Here, the authors review ongoing efforts towards realizing PAM-free nucleases, address potential consequences of eliminating PAM recognition, and propose an alternative nuclease repertoire covering all possible PAM sequences.

Family 1 glycosidases (GH1) are present in the three domains of life and share classical TIM-barrel fold. Structural and biochemical analyses of a resurrected ancestral GH1 enzyme reveal heme binding, not known in its modern descendants. Heme rigidifies the TIM-barrel and allosterically enhances catalysis.

The PAM specificity of SpCas9 can be altered with positive selection during directed evolution. Here the authors use simultaneous positive and negative selection to improve activity on NAG PAMs while reducing activity on NGG PAMs.

Precisely tuning the genetic response to environmental stimuli is a key step in engineering synthetic biology systems. Here, the authors profile 8269 IPTG-induced promoters to deconstruct the relationship between sequence architecture and gene expression.

Nonribosomal lipopeptides contain an acyl chain important for bioactivity, but its incorporation into the peptidyl backbone, mediated by the starter condensation (Cs) domain of nonribosomal peptide synthases, is not fully understood. Here, the authors show that acyl chains of different lengths can be obtained by engineering Cs domains and identify residues that determine the selectivity for acyl chains.

Existing optogenetic methods to induce calcium mobilisation lack selectivity and specificity. Here, the authors design and engineer a single-component light-operated calcium channel to provide optical control over calcium signals and calcium-dependent physiological responses: LOCa.

Citrullination of arginine is crucial for several physiological processes. Here the authors report the site-specific incorporation of citrulline into proteins in mammalian cells using an engineered tRNA synthetase/tRNA pair and a photocaged-citrulline.

In vivo mutagenesis systems can often show restricted capabilities and deleterious off-site mutations. Here the authors fuse base deaminases to T7 RNA polymerase to mutate a target sequence and use dCas9 to define the boundaries of the diversified DNA.

Synthetic biology will transform how we grow food, what we eat, and where we source materials and medicines. Here I have selected six products that are now on the market, highlighting the underlying technologies and projecting forward to the future that can be expected over the next ten years.

A key security challenge with biosecurity threats is determining the responsible actor. In this Perspective, the authors review recent developments in using genetic sequence to assign a lab-of-origin and the potential protection it provides against misuse of synthetic biology.

Large-scale meat production can have negative impacts on public health, the environment and animal welfare. In this Review, the authors consider plant-based and cell-based approaches to meat production and the challenges they face.

The potential for accidental or deliberate misuse of biotechnology is of concern for international biosecurity. Here the authors apply machine learning to DNA sequences and associated phenotypic data to facilitate genetic engineering attribution and identify country-of-origin and ancestral lab of engineered DNA sequences.

Bikaverin is a fungal-derived tetracyclic polyketide with antibiotic, antifungal and anticancer properties. Here, the authors employ various pathway engineering strategies to achieve high level production of bikaverin in baker’s yeast.

Advances in our ability to manipulate genetics leads to deeper understanding of biological systems. In this Perspective, the authors argue that synthetic genomics facilitates complex modifications that open up new areas of research.

Characterizing the assembly of the nuclear pore complex (NPC) remains challenging. Here, the authors develop a set of nanobodies that recognize seven constituent nucleoporins, study their binding characteristics, and apply them to probe accessible and obstructed NPC surfaces in yeast.

The authors have previously reported split-GFP-based contact site sensors (SPLICS) to document endoplasmic reticulum/mitochondria contact sites. Here they extend this work and develop a range of improved SPLICS sensors to detect single and multiple organelle contact sites at different distances.

The success of protein evolution is dependent on the sequence context mutations are introduced into. Here the authors present UMIC-seq that allows consensus generation for closely related genes by using unique molecular identifiers linked to gene variants.

The COVID-19 pandemic has highlighted the need for user-friendly diagnostic techniques. Here, the authors present SHINE, a streamlined and optimised Cas13-based method with accompanying smartphone app for visual diagnosis.

Large volumes of true random numbers are needed for increasing requirements of secure data encryption. Here the authors use the stochastic nature of DNA synthesis to obtain millions of gigabytes of unbiased randomness.

Automated design tools and tailored subunits are beneficial in fine-tuning all components of a complex genetic circuit. Here the authors create E. coli and B. subtilis promoter libraries using FACS and HTS, from which an online promoter design tool has been developed using CNN.