Molecular engineering

  • Article
    | Open Access

    The treatment of snakebite envenoming is currently suboptimal. Existing antivenoms often lack efficacy and may cause adverse reactions. Here, the authors derive, develop, and demonstrate the utility of toxin-specific broadly-neutralizing human monoclonal antibodies with established reactivity across related venom toxins from different snake species and show efficacy in rodent models.

    • Line Ledsgaard
    • , Jack Wade
    •  & Aneesh Karatt-Vellatt
  • Article
    | Open Access

    Genome editing in bacteria normally requires efficient recombination and high transformation efficiencies, which often isn’t. Here the authors report that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive editing.

    • Daphne Collias
    • , Elena Vialetto
    •  & Chase L. Beisel
  • Article
    | Open Access

    The currently available transgenic T cell receptor (TCR) models represent high affinity antigen-TCR interactions. Authors here present an alternative approach to target an exogenous TCR into the physiological Trac locus in the germline of mice, which uncovers that the natural genomic context for TCRs can enhance the antigen sensitivity of lower affinity TCRs and enables the physiologic range of antigen-TCR interaction and a gene dosage dependent mechanism of central tolerance.

    • Meagan R. Rollins
    • , Jackson F. Raynor
    •  & Ingunn M. Stromnes
  • Article
    | Open Access

    RNA provides a unique readout of a cell’s identity, physiologic status, and phenotype. Here the authors deliver an RNA sensing system that can use the information contained within cellular RNA to selectively control the activity of genetic programs.

    • Lauren Gambill
    • , August Staubus
    •  & James Chappell
  • Article
    | Open Access

    Properties of cytidine and adenosine deaminases lead to off-target effects for cytosine base editors (CBEs) and adenine base editors (ABEs). Here the authors report that 25 TadA orthologs could be engineered to generate functional ABEs, CBEs or ACBEs via single/double mutations with minimised off-targets.

    • Shuqian Zhang
    • , Bo Yuan
    •  & Tian-Lin Cheng
  • Article
    | Open Access

    Hypercompact CRISPR-Cas12f systems have been engineered to generate miniABEs but these have limitations. Here the authors generate Cas12f-derived miniCBEs and develop miniABEs with improved editing and targeting scopes; they use these to correct pathogenic mutations in cell lines and introduce mutations in vivo.

    • Shuqian Zhang
    • , Liting Song
    •  & Tian-Lin Cheng
  • Article
    | Open Access

    CRISPR/Cas9-based homing gene drives have emerged as a potential new approach to mosquito control. Here the authors use transgenic lines with germline-specific regulatory elements to express Cas9 and achieve up to 94% inheritance bias, closing the gap between A. aegyptidrives and the highly efficient drives observed in Anopheles species.

    • Michelle A. E. Anderson
    • , Estela Gonzalez
    •  & Luke Alphey
  • Article
    | Open Access

    CRISPR-based gene-drives can carry the Cas9 and guide RNA (gRNA) components in a single-linked cassette or in separate elements inserted into different genomic loci. Here the authors genetically transform and compare full versus split drives, with the former performing less efficiently than predicted.

    • Gerard Terradas
    • , Jared B. Bennett
    •  & Ethan Bier
  • Article
    | Open Access

    Aromatic amino acids in proteins support ligand binding and protein stability. To parse the physiocochemical roles of aromatic interactions, here Galles, Infield and co-authors identify pyrrolysine-based aminoacyl-tRNA synthetases that enable the encoding of fluorinated phenylalanine amino acids.

    • Grace D. Galles
    • , Daniel T. Infield
    •  & Christopher A. Ahern
  • Article
    | Open Access

    Antibiotic and anti-cancer therapy are challenged by mutation-mediated treatment resistance despite many mutations being maladaptive. Here, the authors introduce a system that shows how the probability of the long-term persistence of drug-resistant mutant lineages can be increased in dense microbial populations by acquiring multiple mutations.

    • Serhii Aif
    • , Nico Appold
    •  & Jona Kayser
  • Article
    | Open Access

    The development of broad-spectrum antivirals is an important part of pandemic preparedness and response. Here the authors present ALICE, synthetic biology designer immune-like cells that act as a sense-and-destroy antiviral system can detect viruses from seven different genera, mimicking the human innate immune system.

    • Yidan Wang
    • , Ying Xu
    •  & Haifeng Ye
  • Article
    | Open Access

    Identifying the designers of engineered biological sequences would help promote biotechnological innovation while holding designers accountable. Here the authors present the winners of a 2020 data-science competition which improved on previous attempts to attribute plasmid sequences.

    • Oliver M. Crook
    • , Kelsey Lane Warmbrod
    •  & William J. Bradshaw
  • Article
    | Open Access

    Gene activation methods are valuable for studying gene functions and may have potential applications in bioengineering and medicine. Here the authors developed Narta technology to achieve gene activation by recruiting artificial transcription factors to transcription sites through nascent RNAs of the target gene.

    • Ying Liang
    • , Haiyue Xu
    •  & Baohui Chen
  • Article
    | Open Access

    Engineering enzymes to accept noncanonical cofactor biomimetics is difficult. Here, the authors establish a self-sufficient growth selection method and demonstrate its application in engineering the Lactobacillus pentosus NADH oxidase to efficiently recycle reduced nicotinamide mononucleotide (NMNH).

    • Edward King
    • , Sarah Maxel
    •  & Han Li
  • Article
    | Open Access

    In this work the authors provide a computational workflow for the parallel, from scratch, design of proteins to rapidly explore the shape diversity of protein folds.

    • Thomas W. Linsky
    • , Kyle Noble
    •  & Eva-Maria Strauch
  • Article
    | Open Access

    Reductive stress, reflected by the elevated intracellular NADH/NAD+ ratio, is associated with multiple human diseases. Here, the authors develop a genetic tool to manipulate the ratios of cellular NADH/NAD+ and NADPH/NADP+, and identify purine biosynthesis as an NADH-sensing pathway to mediate reductive stress.

    • Ronghui Yang
    • , Chuanzhen Yang
    •  & Binghui Li
  • Article
    | Open Access

    RNA viruses have been responsible for large-scale epidemics and pandemics throughout the last few centuries. Here, the authors show the design, synthesis and screening of artificial RNA endonuclease XNAzymes capable of cleaving genomic SARS-CoV-2 RNA and self-assembling into enzymatic nanostructures inhibiting cellular viral replication.

    • Pehuén Pereyra Gerber
    • , Maria J. Donde
    •  & Alexander I. Taylor
  • Article
    | Open Access

    A challenge in synthetic biology is the empirical characterisation of genetic parts. Here the authors present FPCountR, a validated method and accompanying R package that enables the precise quantification of fluorescent protein reporters per bacterial cell to be enumerated in ‘proteins per cell’ or nanomolar units without requiring protein purification.

    • Eszter Csibra
    •  & Guy-Bart Stan
  • Article
    | Open Access

    Chimeric antigen receptors (CAR) are a promising option for cell-based immunotherapy for cancer and other immune diseases. Here the authors develop speedingCARs, an integrated CAR design and screening platform based on modular signaling domain shuffling and single cell transcriptomic analyses, and test its potential for identifying and validating novel CAR designs.

    • Rocío Castellanos-Rueda
    • , Raphaël B. Di Roberto
    •  & Sai T. Reddy
  • Article
    | Open Access

    The induction of long-term systemic immunosurveillance can protect against post-surgery tumor recurrence. Here the authors describe the design of optogenetic-controlled cytokine secreting (IFN-β, TNF-α, and IL-12) engineered mesenchymal stem cells loaded into a hydrogel scaffold, eliciting long-term immune memory and preventing post-operative recurrence in preclinical cancer models.

    • Yuanhuan Yu
    • , Xin Wu
    •  & Haifeng Ye
  • Perspective
    | Open Access

    Long-duration human space travel creates challenges for maintaining healthy diets. Here the authors discuss using synthetic biology approaches to modify yeast into an optimal, and enjoyable, food production platform.

    • Briardo Llorente
    • , Thomas C. Williams
    •  & Ian T. Paulsen
  • Article
    | Open Access

    G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. Here the authors, using heterologous GPCR expression and endogenous ligand production, enable synthetic mating in haploid yeast, and GPCR-mediated biosensing in diploid probiotic yeast.

    • Emil D. Jensen
    • , Marcus Deichmann
    •  & Michael K. Jensen
  • Article
    | Open Access

    The immunoglobulin domain framework of antibodies has been a long standing design challenge. Here, the authors describe design rules for tailoring these domains and show they can be accurately designed, de novo, with high stability and the ability to scaffold functional loops.

    • Tamuka M. Chidyausiku
    • , Soraia R. Mendes
    •  & Enrique Marcos
  • Article
    | Open Access

    Applications of the SCRaMbLE process are hindered due to the lack of facile and tight regulation and limited understanding of key factors that may affect the rearrangement outcomes. Here the authors present an approach to precisely regulate SCRaMbLE recombination in a dose-dependent manner using genetic code expansion technology with low basal activity.

    • Huiming Zhang
    • , Xian Fu
    •  & Yue Shen
  • Article
    | Open Access

    “Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. Here the authors show that PopZ condensate dynamics support cell division and using PopZ modular architecture, the tunable PopTag platform was developed to enable designer condensates.”

    • Keren Lasker
    • , Steven Boeynaems
    •  & Lucy Shapiro
  • Article
    | Open Access

    Computational properties of neuronal networks have been applied to computing systems using simplified models comprising repeated connected nodes. Here the authors create layered assemblies of genetically encoded devices that perform non-binary logic computation and signal processing using combinatorial promoters and feedback regulation.

    • Luna Rizik
    • , Loai Danial
    •  & Ramez Daniel
  • Article
    | Open Access

    DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. Here the authors present a strand assembly algorithm (DBGPS) using de Bruijn graph and greedy path search.

    • Lifu Song
    • , Feng Geng
    •  & Ying-Jin Yuan
  • Article
    | Open Access

    Arising through multiple binding elements, multivalency can specify the avidity, duration, cooperativity, and selectivity of biomolecular interactions, but quantitative prediction and design of these properties has remained challenging. Here the authors enable facile analysis and engineering of multivalent binding by developing MVsim, a simulator that incorporates biochemical and biophysical parameters of interacting molecules and is accessible through a graphical user interface.

    • Bence Bruncsics
    • , Wesley J. Errington
    •  & Casim A. Sarkar
  • Article
    | Open Access

    CRISPR gene activation and inhibition has become a powerful synthetic tool for influencing the expression of native genes for foundational studies, cellular reprograming, and metabolic engineering. Here the authors demonstrate near leak-free, inducible expression of a polycistronic array containing up to 24 gRNAs from two orthogonal CRISPR/Cas systems.

    • William M. Shaw
    • , Lucie Studená
    •  & Rodrigo Ledesma-Amaro
  • Article
    | Open Access

    De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build enzymes for efficient energy conversion. Here, the authors develop an artificial photosynthetic reaction center that functions without the need for sacrificial electron donors or acceptors.

    • Nathan M. Ennist
    • , Zhenyu Zhao
    •  & Christopher C. Moser
  • Article
    | Open Access

    Toe-hold-mediated strand displacement (DSD) is a widely used molecular tool in applications such as DNA computing and nucleic acid diagnostics. Here the authors characterize dozens of orthogonal barcode sequences that can be used for monitoring the output kinetics of multiplexed DSD reactions in real-time using a commercially-available portable nanopore array device.

    • Karen Zhang
    • , Yuan-Jyue Chen
    •  & Jeff Nivala
  • Article
    | Open Access

    Base editing is promising for gene therapy, but in vivo delivery has been limiting. Here the authors perform structure-based rational engineering of the cytosine base editing system Target-AID to minimise off-target effects and decrease its size.

    • Ang Li
    • , Hitoshi Mitsunobu
    •  & Keiji Nishida
  • Article
    | Open Access

    In vitro library screening is a powerful approach to identify functional biopolymers, but only covers a fraction of possible sequences. Here, the authors use experimental in vitro selection results to train a conditional variational autoencoder machine learning model that generates biopolymers with no apparent sequence similarity to experimentally derived examples, but that nevertheless bind the target molecule with similar potent binding affinity.

    • Jonathan C. Chen
    • , Jonathan P. Chen
    •  & David R. Liu
  • Article
    | Open Access

    Whole-genome recoding has been shown to enable nonstandard amino acids, biocontainment and viral resistance in bacteria. Here the authors extend this to human cells using base editing to convert TAG to TAA for 33 essential genes via a single transfection followed by examining base-editing genome-wide.

    • Yuting Chen
    • , Eriona Hysolli
    •  & George Church
  • Article
    | Open Access

    The invention of the Fourier integral in the 19th century laid the foundation for modern spectral analysis methods. Here the authors develop frequency-based methods for analyzing the reaction mechanisms within living cells from distinctively noisy single-cell output trajectories and present forward engineering of synthetic oscillators and controllers.

    • Ankit Gupta
    •  & Mustafa Khammash
  • Article
    | Open Access

    Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. Here the authors present a scarless, biallelic, and 100 kb-scale mutagenesis in human cells that uncovers functional significances of endogenous introns and retrotransposons in the chromatin context.

    • Tomoyuki Ohno
    • , Taichi Akase
    •  & Yasunori Aizawa
  • Article
    | Open Access

    Site-specific recombination (SSR) is an important tool in synthetic biology, but its applications are limited by the inability to predictably tune SSR reaction rates. Here, using quantitative high-throughput experiments and machine learning, the authors achieve rational control of a DNA attachment site sequence to predictably modulate site-specific recombination rates both in vitro and in cells.

    • Qiuge Zhang
    • , Samira M. Azarin
    •  & Casim A. Sarkar
  • Article
    | Open Access

    The range of available copy numbers for cloning vectors is largely restricted to the handful of ORIs that have been isolated from plasmids found in nature. Here the authors introduce a plasmid system that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell.

    • Miles V. Rouches
    • , Yasu Xu
    •  & Guillaume Lambert
  • Article
    | Open Access

    Bacteroides species are prominent members of the human gut microbiota. Here the authors develop a platform technology (consortium transcriptional programming) in five human donor Bacteroides chassis cells—enabling complex decision-making within said community, which can be used for the development of living therapeutics.

    • Brian D. Huang
    • , Thomas M. Groseclose
    •  & Corey J. Wilson
  • Article
    | Open Access

    Clostridioides difficile infection (CDI) results in significant morbidity and mortality in hospitalised patients. Here the authors engineer probiotics to restore intestinal bile salt metabolism in response to antibiotic-induced microbiome dysbiosis significantly inhibit Clostridioides difficile infection in model mice, presenting a microbiome-based antimicrobial strategy

    • Elvin Koh
    • , In Young Hwang
    •  & Matthew Wook Chang
  • Article
    | Open Access

    Optimising antibody properties such as affinity can be detrimental to other key properties. Here the authors use machine learning to simplify the identification of antibodies with co-optimal levels of affinity and specificity for a clinical-stage antibody that displays high levels of on- and off-target binding.

    • Emily K. Makowski
    • , Patrick C. Kinnunen
    •  & Peter M. Tessier
  • Article
    | Open Access

    The bacterial respiratory electron transport system (ETS) is branched to allow condition-specific modulation of energy metabolism. Here the authors examine the systems level properties of aerobic electron transport system using adaptive laboratory evolution and multi-omics analyses.

    • Amitesh Anand
    • , Arjun Patel
    •  & Bernhard O. Palsson