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| Open AccessSpyMask enables combinatorial assembly of bispecific binders
Bispecific antibody architecture is often important for function but rarely optimized. Here, authors present a modular approach to assemble bispecifics in varied formats using a SpyTag/SpyCatcher approach called SpyMask, and build anti-HER2 bispecifics whose activities depend on binder orientation and bispecific geometry.
- Claudia L. Driscoll
- , Anthony H. Keeble
- & Mark R. Howarth
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Article
| Open AccessCRISPR-powered quantitative keyword search engine in DNA data storage
Targeting the files containing content-of-interest is a challenge in DNA data storage. Here, the authors develop a CRISPR-powered search engine to quantitatively identify the keyword in files stored in DNA.
- Jiongyu Zhang
- , Chengyu Hou
- & Changchun Liu
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Article
| Open AccessAdvancing the scale of synthetic biology via cross-species transfer of cellular functions enabled by iModulon engraftment
Machine learning applied to large compendia of transcriptomic data has enabled the decomposition of bacterial transcriptomes to identify independently modulated sets of genes. Here the authors present iModulon-based engineering for precise identification of genes for cross-species function transfer to streamline synthetic biology for strain development and biomanufacturing.
- Donghui Choe
- , Connor A. Olson
- & Bernhard O. Palsson
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Article
| Open AccessEdible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit
Fungi have the potential to produce sustainable foods for a growing population, but current products are based on a small number of strains with inherent limitations. Here, the authors develop genetic tools for an edible fungus and engineer its nutritional value and sensory appeal for alternative meat applications.
- Vayu Maini Rekdal
- , Casper R. B. van der Luijt
- & Jay D. Keasling
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Article
| Open AccessRepurposing CRISPR-Cas13 systems for robust mRNA trans-splicing
The ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, the authors demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT).
- David N. Fiflis
- , Nicolas A. Rey
- & Aravind Asokan
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Article
| Open AccessPHEIGES: all-cell-free phage synthesis and selection from engineered genomes
Bacteriophages have great potential in both medicine and biotechnology. Here the authors present PHEIGES, a cell-free method for phage genome engineering, synthesis and selection based on T7, which allows direct selection of engineered and mutant phages without compartmentalization.
- Antoine Levrier
- , Ioannis Karpathakis
- & Vincent Noireaux
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Article
| Open AccessCas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements
Engineered biosensing bacteria can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. Here the authors present a robust biocontainment assisted by Cas9 and an engineered gene expression control combined in a genetically engineered human commensal bacterium that successfully functioned in a mouse intestinal tract as well as cell culture condition.
- Naoki Hayashi
- , Yong Lai
- & Timothy K. Lu
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Article
| Open AccessBiosensor and machine learning-aided engineering of an amaryllidaceae enzyme
Amaryllidaceae alkaloids, such as the Alzheimer’s medication galantamine, are currently extracted from low-yielding daffodils. Here, authors pair biosensor-assisted screening with machine learning-guided protein design to rapidly engineer an improved Amaryllidaceae enzyme in a microbial host.
- Simon d’Oelsnitz
- , Daniel J. Diaz
- & Andrew D. Ellington
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Article
| Open AccessMachine learning-aided design and screening of an emergent protein function in synthetic cells
Here, the authors introduce a pipeline to screen machine learning generated variants of a protein that forms intracellular spatiotemporal patterns in E. coli, demonstrating the best variants can substitute the wildtype gene.
- Shunshi Kohyama
- , Béla P. Frohn
- & Petra Schwille
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Article
| Open AccessMotility of an autonomous protein-based artificial motor that operates via a burnt-bridge principle
Inspired by biology, great progress has been made in creating artificial molecular motors. Here the authors report the synthesis and characterization of the Lawnmower, an autonomous, protein-based artificial molecular motor and show their design is capable of track-guided motion.
- Chapin S. Korosec
- , Ivan N. Unksov
- & Nancy R. Forde
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Article
| Open AccessOrthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells
The lack of control over Cas13 activity has limited its utility. Here the authors report Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands (CRISTAL), controlled by orthogonal split inducible Cas13 effectors that can be turned ON or OFF, providing precise temporal control.
- Yage Ding
- , Cristina Tous
- & Wilson W. Wong
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Article
| Open AccessA system for inducible mitochondria-specific protein degradation in vivo
Conditional degradation of proteins is instrumental to advance our knowledge of cell biology but has been lacking for organelles like mitochondria. Here, the authors develop a proteolysis system based on the mycoplasma Lon protease that functions selectively within mitochondria in yeast and human cells.
- Swastika Sanyal
- , Anna Kouznetsova
- & Camilla Björkegren
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Article
| Open AccessOrthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts
Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Here the authors develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation.
- Charlotte Cautereels
- , Jolien Smets
- & Kevin J. Verstrepen
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Article
| Open AccessCombinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast
Fine-tuning the expression of biosynthetic pathway genes is crucial to improve microbial production titres. Here, the authors present GEMbLeR, an optimization strategy to balance the expression of multiple genes simultaneously over a wide range.
- Charlotte Cautereels
- , Jolien Smets
- & Kevin J. Verstrepen
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Article
| Open AccessAAV-delivered muscone-induced transgene system for treating chronic diseases in mice via inhalation
Long-term control of therapeutic transgene expression is needed. Here the authors report a muscone-induced transgene system packaged into AAVs based on a G protein-coupled murine olfactory receptor and a synthetic cAMP-responsive promoter: they show dose- and exposure-time-dependent gene expression control in mice.
- Xin Wu
- , Yuanhuan Yu
- & Haifeng Ye
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Article
| Open AccessLogical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes
Descriptive data in biomedical research are expanding rapidly, but functional validation methods lag behind. Here, authors present Logical Synthetic cis-regulatory DNA, a framework to design reporters that mark cellular states and pathways, showcasing its applicability to complex phenotypic states.
- Carlos Company
- , Matthias Jürgen Schmitt
- & Gaetano Gargiulo
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Article
| Open AccessAnti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages
CRISPR-based gene drives have the potential to spread within populations and are considered as promising vector control tools. Here the authors show an anti-drive mosquito strain that prevents the spread and collapse of a population suppression gene drive in laboratory Anopheles mosquito large cage trials in complex ecological and behavioral conditions.
- Rocco D’Amato
- , Chrysanthi Taxiarchi
- & Ruth Müller
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Article
| Open AccessLarge-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae
Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. Here the authors present an engineered yeast strain with 83 sparsely distributed loxPsym sites across the genome can genrerate large-scale genomic rearrangements, which benefits cell fitness under stress and boosts the SCRaMbLE system when combined with synthetic chromosomes.
- Li Cheng
- , Shijun Zhao
- & Junbiao Dai
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Article
| Open AccessA multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations
Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Here the authors find that a CRISPR/Cas9 based split gene drive in Aedes aegypti could successfully bias inheritance up to 89% over successive generations in a multi-cage trial with further deep sequencing suggesting that the multiplexing design could mitigate resistance allele formation.
- Michelle A. E. Anderson
- , Estela Gonzalez
- & Luke Alphey
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Article
| Open AccessRapid and visual identification of β-lactamase subtypes for precision antibiotic therapy
The rapid identification of drug-resistant bacteria is vital for effective treatment and to avoid antibiotic misuse. Here authors report a paper-based sensor which utilises chromogenic carbapenem and cephalosporin substrates for the identification and discrimination of β-lactamase subtypes.
- Wenshuai Li
- , Jingqi Li
- & Dingbin Liu
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Article
| Open AccessProgrammable RNA base editing with photoactivatable CRISPR-Cas13
Cas13 systems suffer from a lack of spatiotemporal control. Here the authors report paCas13, a light-inducible Cas13 system created by fusing Magnet with fragment pairs; they also report padCas13, a light-inducible base-editing system by fusing ADAR2 to catalytically inactive paCas13 fragments.
- Jeonghye Yu
- , Jongpil Shin
- & Won Do Heo
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Comment
| Open AccessA bumpy road ahead for genetic biocontainment
While the research community continues to develop novel proposals for intrinsic biocontainment of genetically engineered organisms, translation to real-world deployment faces several challenges.
- Dalton R. George
- , Mark Danciu
- & Emma K. Frow
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Article
| Open Accessi-shaped antibody engineering enables conformational tuning of biotherapeutic receptor agonists
In contrast to their clinical success as inhibitors and targeting agents, antibodies have generally been ineffective as receptor agonists. Here, Romei et al. leverage a natural homotypic interface to tune antibody geometry, enabling optimization of agonist activity for multiple therapeutic targets.
- Matthew G. Romei
- , Brandon Leonard
- & Greg A. Lazar
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Article
| Open AccessGene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata
Homing-based gene drives are novel interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here the authors demonstrate the feasibility of a gene drive approach for the genetic control of the agricultural pest, the medfly, based on complete female-to-male sex conversion.
- Angela Meccariello
- , Shibo Hou
- & Nikolai Windbichler
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Article
| Open AccessCharacterizations of a neutralizing antibody broadly reactive to multiple gluten peptide:HLA-DQ2.5 complexes in the context of celiac disease
Targeting gluten antigens presents a plausible therapy option for celiac disease. Here the authors generate and characterize a broadly neutralizing antibody recognizing more than 25 gluten peptide:HLA-DQ2.5 complexes, with structural analyses implicating its mode of interaction, and with mouse in vivo studies supporting its therapeutic feasibility.
- Yuu Okura
- , Yuri Ikawa-Teranishi
- & Tomoyuki Igawa
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Article
| Open AccessBuilding synthetic chromosomes from natural DNA
Building synthetic chromosomes from natural components is an unexplored alternative to de novo chromosome synthesis that may have many potential applications. In this paper, the authors report CReATiNG, a method for constructing synthetic chromosomes from natural components in yeast.
- Alessandro L. V. Coradini
- , Christopher Ne Ville
- & Ian M. Ehrenreich
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Article
| Open AccessOptimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors
Designing optogenetically functionalized G protein-coupled receptors (optoXRs) to mimic endogenous signaling in vivo is challenging. Here, the authors optimize the design of optoXRs by considering evolutionary conserved protein interactions, and they employ this approach in fruit flies.
- Fangmin Zhou
- , Alexandra-Madelaine Tichy
- & Peter Soba
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Review Article
| Open AccessCustomizing cellular signal processing by synthetic multi-level regulatory circuits
As synthetic biology permeates society, the signal processing circuits in engineered living systems must be customized to meet practical demands. In this review, the authors outline design strategies for the DNA, RNA, and protein-level circuits and the hybrid “multi-level” circuits.
- Yuanli Gao
- , Lei Wang
- & Baojun Wang
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Article
| Open AccessEngineered immunogens to elicit antibodies against conserved coronavirus epitopes
A pan-betacoronavirus vaccine will likely require the elicitation of antibodies against spike regions conserved across diverse coronaviruses. Here, authors computationally engineer and experimentally validate immunogens to elicit antibodies against two such spike regions.
- A. Brenda Kapingidza
- , Daniel J. Marston
- & Mihai L. Azoitei
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Article
| Open AccessInterrogating ligand-receptor interactions using highly sensitive cellular biosensors
The interaction of membrane-resident proteins plays an essential role in biological processes. Here the authors describe cellular biosensors based on chimeric receptors, as a tool to study the interaction of receptor-ligand pairs such as immune checkpoint molecules or virus attachment proteins and their receptors.
- Maximilian A. Funk
- , Judith Leitner
- & Peter Steinberger
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Article
| Open AccessA simeprevir-inducible molecular switch for the control of cell and gene therapies
Chemical inducer of dimerization (CID) modules can be used to effectively control biological processes; however, CID modules have been explored primarily in engineering cells for in vitro applications using inducers that have limited clinical utility. Here, the authors identify a CID module with favorable properties to enable rapid translation from in vitro applications to potential use in humans.
- Stacey E. Chin
- , Christina Schindler
- & Natalie J. Tigue
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Article
| Open AccessCRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus
Culex mosquitoes are carriers of major diseases like West Nile virus and are a public health concern. Here the authors present a CRISPR-Cas9 gene drive as a control technology in the Culex quinquefasciatus mosquito species.
- Tim Harvey-Samuel
- , Xuechun Feng
- & Valentino M. Gantz
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Article
| Open AccessDiurnal switches in diazotrophic lifestyle increase nitrogen contribution to cereals
Engineering ammonium excretion diazotrophs suffers from severe penalties to the bacteria. Here, the authors utilize a thermo-sensitive glutamine synthetase-based regulatory switch that permits diurnal changes in diazotrophic lifestyle, coincident with seasonal temperatures for cereal cultivation.
- Yuqian Tang
- , Debin Qin
- & Yi-Ping Wang
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Article
| Open AccessEngineering sulfonate group donor regeneration systems to boost biosynthesis of sulfated compounds
Sufficient supply of sulfonate group donor is critical to biomanufacturing of the sulfate containing compounds. Here, the authors engineer two sulfonate group donor regeneration systems, including 3'-phosphoadenosine-5'-phosphosulfate and the newly discovered 5'-phosphosulfate, to boost biosynthesis of sulfated compounds.
- Ruirui Xu
- , Weijao Zhang
- & Zhen Kang
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Article
| Open AccessRational design of microRNA-responsive switch for programmable translational control in mammalian cells
Artificial regulation of translation by intracellular RNAs has many potential applications. Here, authors design a platform capable of miRNA-triggered upregulation or downregulation using a single RNA construct, and demonstrate its use in constructing logic gates and cell-type classifiers.
- Hui Ning
- , Gan Liu
- & Zhen Xie
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Article
| Open AccessCell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides
Deep learning holds a great promise for the discovery and design of bioactive peptides, but experimental approaches to validate candidates in high throughput and at low cost are needed. Here, the authors combine deep learning and cell free biosynthesis for antimicrobial peptide (AMP) development and identify 30 functional AMPs, of which six with broad-spectrum activity against drug-resistant pathogens.
- Amir Pandi
- , David Adam
- & Tobias J. Erb
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Article
| Open AccessRemodeling the cellular stress response for enhanced genetic code expansion in mammalian cells
Genetic code expansion (GCE) is a protein engineering tool that enables programmed and site-specific installation of noncanonical amino acids into proteins. Here, authors show that cellular stress remodelling boosts GCE in mammalian cells including GCE realized by orthogonally translating organelles.
- Mikhail E. Sushkin
- , Christine Koehler
- & Edward A. Lemke
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Article
| Open AccessLactate biosensors for spectrally and spatially multiplexed fluorescence imaging
l-Lactate is increasingly recognized as a key metabolite and signalling molecule in mammals, but the methods to investigate it in vivo have been limited. Here, authors report a pair of improved biosensors—one green and one red—for visualizing l-lactate both inside and outside of cells.
- Yusuke Nasu
- , Abhi Aggarwal
- & Robert E. Campbell
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Article
| Open AccessRNA-based translation activators for targeted gene upregulation
Many diseases are driven by the insufficient expression of critical genes, but few technologies are capable of rescuing these endogenous protein levels. Here, Cao et al. present an RNA-based technology that boosts protein production from endogenous mRNAs by upregulating their translation.
- Yang Cao
- , Huachun Liu
- & Bryan C. Dickinson
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Article
| Open AccessCircular single-stranded DNA as switchable vector for gene expression in mammalian cells
Synthetic gene networks in mammalian cells are currently limited to either transcription factors or RNA regulators. Here, the authors develop a regulatory approach based on circular single-stranded DNA, which can be used as a conformationally switchable genetic vector in mammalian cells.
- Linlin Tang
- , Zhijin Tian
- & Jie Song
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Article
| Open AccessBiosynthesis and engineering of the nonribosomal peptides with a C-terminal putrescine
Nonribosomal peptides have diverse bioactivities and can possess unusual moieties at their C-terminus, such as polyamines. In this study, the authors identify a class of dodecapeptides glidonins that feature diverse N-terminal modifications and a uniform putrescine moiety at the C-terminus, elucidate their biosynthesis, and introduce the putrescine into the C-terminus of other nonribosomal peptides.
- Hanna Chen
- , Lin Zhong
- & Xiaoying Bian
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Article
| Open AccessSonogenetic control of multiplexed genome regulation and base editing
Exogenous control of genes in vivo is important. Here the authors report a system that can be inducibly activated through thermal energy produced by ultrasound absorption and use this to control induction of gene activation and base editing: they apply this in cell lines and in a mouse model.
- Pei Liu
- , Josquin Foiret
- & Lei S. Qi
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Article
| Open AccessA robust yeast biocontainment system with two-layered regulation switch dependent on unnatural amino acid
Synthetic auxotrophy in which cell viability depends on the presence of an unnatural amino acid provides a powerful strategy to restrict unwanted propagation of genetically modified organisms in open environments and potentially prevent industrial espionage. Here the authors establish a general framework for the creation and optimization of synthetic auxotrophs in yeast.
- Tiantian Chang
- , Weichao Ding
- & Xian Fu
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Article
| Open AccessDigital data storage on DNA tape using CRISPR base editors
DNA is an alternative to data storage materials for its durability, density, and energetics. Here the authors demonstrate the storage of digital information on DNA molecules using base-editing.
- Afsaneh Sadremomtaz
- , Robert F. Glass
- & Reza Zadegan
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Article
| Open AccessNext-generation CRISPR gene-drive systems using Cas12a nuclease
One method for reducing the impact of vector-borne diseases is through the use of CRISPR-based gene drives, which manipulate insect populations due to their ability to rapidly propagate desired genetic traits into a target population. Here the authors describe a Cas12a gene drive system whose activity can be finetuned in a temperature-dependent manner.
- Sara Sanz Juste
- , Emily M. Okamoto
- & Víctor López Del Amo
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Article
| Open AccessDeep flanking sequence engineering for efficient promoter design using DeepSEED
Designing promoters with desired properties is crucial in synthetic biology. Here, authors introduce DeepSEED, an AI-aided flanking sequence optimisation framework which combines expert knowledge with deep learning techniques to efficiently design promoters in both eukaryotic and prokaryotic cells.
- Pengcheng Zhang
- , Haochen Wang
- & Xiaowo Wang
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Article
| Open AccessNanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera
Most current anti-coronavirus nanoparticle vaccines target epitopes within the RBD. Here, the authors developed nanoparticles displaying an array of spike fusion proteins derived from various coronaviruses and show that immunizing mice with these vaccines elicits broad and potent cross-reactive antibodies.
- Geoffrey B. Hutchinson
- , Olubukola M. Abiona
- & Kizzmekia S. Corbett-Helaire
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Article
| Open AccessA digital twin for DNA data storage based on comprehensive quantification of errors and biases
Archiving data in synthetic DNA offers unprecedented storage density and longevity. To understand how experimental choices affect the integrity of digital data stored in DNA, the authors study the evolution of errors and bias and with a digital twin they supply tools for experimental planning and design of error-correcing codes.
- Andreas L. Gimpel
- , Wendelin J. Stark
- & Robert N. Grass
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Comment
| Open AccessEngineered and natural gene drives: mechanistically the same, yet not same in kind
We propose the use of the terms natural gene drive (NGD) and engineered gene drive (EGD) arguing against James et al.
1 , who think both should be included within the term “gene drive”, based on their mechanistic similarities.- Raul F. Medina
- & Jennifer Kuzma