Synthetic Biology

BRAIDing receptors for cell specific targeting

1. Hui Chen
2. Sung-Jin Lee
3. Ryan Li
4. Asmiti Sura
5. Nicholas Suen
6. Archana Dilip
7. Yan Pomogov
8. Meghah Vuppalapaty
9. Chenggang Lu
10. Yorick Post
11. Yang Li

• Curated by eLife

  eLife assessment

  This important study introduces a new therapeutic cell targeting system, where an active molecule is divided into inactive or less active parts that are subsequently linked via a bridging receptor on a target cell. Solid experimental proof of concept involving the activation of Wnt signaling in hepatocytes is presented, which could be further strengthened by demonstrating the specificity of the effect when applied to a mix of different cell types. This study may be of substantial interest to medical biologists focused on targeted therapeutics or seeking to utilize cell type-specific pathway modulation in their research.

Reviewed by eLife

Effects of growth feedback on gene circuits: A dynamical understanding

1. Ling-Wei Kong
2. Wenjia Shi
3. Xiao-Jun Tian
4. Ying-Cheng Lai

• Curated by eLife

  eLife assessment

  This valuable study focuses on the impact of growth feedback on the performance of artificial gene circuits capable of achieving adaptive responses, an important problem in synthetic biology. Through solid computational analysis, the authors identify specific failure mechanisms, as well as core topologies associated with robust performance based on systematic analysis of over four hundred circuit topologies. The results will be of interest to those working on engineering gene circuits for diverse applications.

Reviewed by eLife

Specific Modulation of CRISPR Transcriptional Activators through RNA-Sensing Guide RNAs in Mammalian Cells and Zebrafish Embryos

1. Oana Pelea
2. Sarah Mayes
3. Quentin RV. Ferry
4. Tudor A. Fulga
5. Tatjana Sauka-Spengler

• Curated by eLife

  eLife assessment

  The authors aim to develop a CRISPR system that can be activated upon sensing an RNA. As an initial step to this goal, they describe RNA-sensing guide RNAs for controlled activation of CRISPR modification. Many of the data look convincing and while several steps remain to achieve the stated goal in an in vivo setting and for robust activation by endogenous RNAs, the current work will be important for many in the field.

Reviewed by eLife

Autotrophic growth of E. coli is achieved by a small number of genetic changes

1. Roee Ben-Nissan
2. Eliya Milshtein
3. Vanessa Pahl
4. Benoit de Pins
5. Ghil Jona
6. Dikla Levi
7. Hadas Yung
8. Noga Nir
9. Dolev Ezra
10. Shmuel Gleizer
11. Hannes Link
12. Elad Noor
13. Ron Milo

• Curated by eLife

  eLife assessment

  This is an important follow-up study to a previous paper in which the authors reconstituted CO2 metabolism in Escherichia coli (autotrophy). Here, the authors define a set of three mutations that promote autotrophy, highlighting the malleability of E. coli metabolism. The authors make a convincing case that mutations in pgi are loss-of-function mutations that prevent metabolic efflux from the reductive pentose phosphate autocatalytic cycle, but claims about the role of mutations in two other genes - crp and rpoB - are currently incomplete. This research will be particularly interesting to synthetic biologists, systems biologists, and metabolic engineers aiming to develop synthetic autotrophic microorganisms.

Reviewed by eLife

Comprehensive mutagenesis maps the effect of all single codon mutations in the AAV2 rep gene on AAV production

1. Nina K. Jain
2. Pierce J. Ogden
3. George M. Church

• Curated by eLife

  eLife assessment

  This study presents a valuable and comprehensive mutagenesis map of the AAV2 rep gene, which will undoubtedly capture the interest of scientists working with adeno-associated viruses and those engaged in the field of gene therapy. The thorough characterization of massive rep variants across multiple AAV production systems bolsters the claims made in the study, highlighting its utility in enhancing our understanding of Rep protein function and advancing gene therapy applications. Despite some limitations, such as the lack of measurements on AAV transduction efficiency, the evidence presented is solid and establishes a strong foundation that will stimulate and inform future research in the field.

Reviewed by eLife

Light inducible protein degradation in E. coli with LOVtag

1. Nathan Tague
2. Cristian Coriano-Ortiz
3. Michael B. Sheets
4. Mary J. Dunlop

• Curated by eLife

  eLife assessment

  This paper describes useful tools for the light-induced degradation of proteins in bacteria, which will be appreciated by the community. The methods and data analyses are solid but the strength of evidence for the tool working as advertised is still incomplete. The study will be of interest to colleagues in the fields of microbiology and synthetic biology.

Reviewed by eLife

Ribozyme-phenotype coupling in peptide-based coacervate protocells

1. Kristian Le Vay
2. Elia Salibi
3. Basusree Ghosh
4. T-Y Dora Tang
5. Hannes Mutschler

• Curated by eLife

  eLife assessment

  Experimental models of simple cell-like compartments can help us to understand how biology operated early in its history. The authors convincingly show how the properties of coacervate droplets can be influenced by the activity of ribozymes inside them. This important result potentially provides a new route for biologists or chemists to establish cell mimics that support the evolution of biomolecules within.

Reviewed by eLife

Macroscopic control of synchronous electrical signaling with chemically-excited gene expression

1. M. García-Navarrete
2. Merisa Avdovic
3. S. Pérez García
4. D. Ruiz Sanchis
5. K. Wabnik

• Curated by eLife

  eLife assessment

  This manuscript will be of interest to those working on non-neuronal bioelectricity, particular synthetic biologists and bioengineers. The primary contribution is the ability to leverage engineered gene circuits to control cellular membrane potential. We find issue, however, with the presentation of the data in this work as electrical communication since the synchronous behavior largely arises from external chemical stimuli.

Reviewed by eLife

Resurrecting essential amino acid biosynthesis in a mammalian cell

1. Julie Trolle
2. Ross M. McBee
3. Andrew Kaufman
4. Sudarshan Pinglay
5. Henri Berger
6. Sergei German
7. Liyuan Liu
8. Michael J. Shen
9. Xinyi Guo
10. J. Andrew Martin
11. Michael Pacold
12. Drew R. Jones
13. Jef D. Boeke
14. Harris H. Wang

• Curated by eLife

  Evaluation Summary:

  In this study, Trolle et al aimed to introduce methionine, threonine, isoleucine, and valine biosynthetic pathways into Chinese Hamster Ovary (CHO) cells. While this was unsuccessful for methionine, threonine, and isoleucine, introduction of valine synthesis rendered CHO cells partially independent on exogenous valine. Although introduction of essential amino acid biosynthetic pathways into mammalian cells is of potentially broad interest to the fields of synthetic biology, biotechnology and metabolism, there were concerns regarding incomplete demonstration that the introduction of valine pathway into CHO cells is sufficient to sustain homeostasis in the absence of exogenous valine. Further metabolic/biochemical characterization of valine-producing CHO cells is warranted.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

Reviewed by eLife

De novo designed transmembrane domains tune engineered receptor functions

1. Assaf Elazar
2. Nicholas J. Chandler
3. Ashleigh S. Davey
4. Jonathan Y. Weinstein
5. Julie V. Nguyen
6. Raphael Trenker
7. Ryan S. Cross
8. Misty R. Jenkins
9. Melissa J. Call
10. Matthew E. Call
11. Sarel J. Fleishman

• Curated by eLife

  Evaluation Summary:

  This is an interesting paper that uses de novo protein design to probe the effects of oligomerization state on the activity of chimeric antigen receptors (CARS). The successful design of transmembrane domains with specific oligomeric states is an impressive result on its own. The proteins were designed using rotamer-based sequence optimization in Rosetta with an energy function specific for the membrane environment. After experimentally evaluating a couple rounds of designs, the investigators settled on a design protocol that also included screening of the design candidates with docking simulations in alternative oligomerization states to check that the sequences preferred the desired oligomerization state. The designs were experimentally evaluated with gel electrophoresis and X-ray crystallography. In the end, designs that adopted well-defined dimers, trimers, or tetramers were created and carried forward in experiments as CARs.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

Reviewed by eLife

Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells

1. Sarah Lensch
2. Michael H. Herschl
3. Connor H. Ludwig
4. Joydeb Sinha
5. Michaela M. Hinks
6. Adi Mukund
7. Taihei Fujimori
8. Lacramioara Bintu

• Curated by eLife

  Evaluation Summary:

  This study describes a novel approach to investigate how the transcriptional repressors KRAB and HDAC4 repress gene expression, how repression spreads over differing genomic distances, and what the role of insulator elements is in blocking the spread of repression and in reactivation of repressed genes. The results of this study allow modeling of the coordinated repression or activation of closely linked genes and should be of wide interest to researchers interested in chromatin and gene expression.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Reviewed by eLife

Measuring the tolerance of the genetic code to altered codon size

1. E. DeBenedictis
2. D. Söll
3. K. Esvelt

• Curated by eLife

  Evaluation Summary:

  Using a phage-based library generation and selection, the authors generated a suite of 4-base decoding tRNAs with improved efficiency in quadruplet decoding. The data represent an important step toward enhancing protein synthesis with 4-base codons. Overall, the approach to generate many tRNA variants with quadruplet anticodons is intriguing and provides a wealth of valuable information to the field. The results, once some of the reviewer concerns have been addressed, should become foundational for the field of synthetic biology.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

Reviewed by eLife

Development and Characterization of PAR-Trackers: New Tools for Detecting Poly(ADP-ribose) In Vitro and In Vivo

1. Sridevi Challa
2. Keun W. Ryu
3. Amy L. Whitaker
4. Jonathan C. Abshier
5. Cristel V. Camacho
6. W. Lee Kraus

• Curated by eLife

  Evaluation Summary:

  Challa and Ryu et al. systematically evaluated various combinations of ADP-ribose-binding modules to make sensors detecting poly(ADP-ribose). They developed and tested two indicator designs optimized for analyses in cell culture (dimerization-dependent GFP-based) or intact tissues (split Nano luciferase-based). Overall, with further experimental controls and quantification, this timely set of cell biology probes will be useful to study the biological functions of ADP-ribosylation in cultured cells and whole organisms.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Reviewed by eLife

Heterogeneity of the GFP fitness landscape and data-driven protein design

1. Louisa Gonzalez Somermeyer
2. Aubin Fleiss
3. Alexander S. Mishin
4. Nina G. Bozhanova
5. Anna A. Igolkina
6. Jens Meiler
7. Maria-Elisenda Alaball Pujol
8. Ekaterina V. Putintseva
9. Karen S. Sarkisyan
10. Fyodor A. Kondrashov

• Curated by eLife

  Evaluation Summary:

  The manuscript dives into how protein structure/function robustness to mutation or polymorphism relates across evolutionary distance. The work indicates that evolutionarily related genes will have different shapes of robustness to variation, and that this will not necessarily track with phylogenetic relationships. The conclusions have potential ramifications for protein engineering, protein structure as well as population genetics and phylogenetics.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by eLife

Resolving phylogenetic and biochemical barriers to functional expression of heterologous iron-sulphur cluster enzymes

1. Helena Shomar
2. Pierre Simon Garcia
3. Elena Fernández-Fueyo
4. Francesca D’Angelo
5. Martin Pelosse
6. Rita Rebelo Manuel
7. Ferhat Büke
8. Siyi Liu
9. Niels van den Broek
10. Nicolas Duraffourg
11. Carol de Ram
12. Martin Pabst
13. Simonetta Gribaldo
14. Beatrice Py
15. Sandrine Ollagnier de Choudens
16. Gregory Bokinsky
17. Frédéric Barras

• Curated by eLife

  Evaluation Summary:

  Three of Nature's life-sustaining processes, respiration, photosynthesis, and nitrogen fixation, all rely on proteins (Fe-S protein) that contain simple inorganic cofactors constructed of Fe and S (Fe-S clusters). Fe-S proteins also participate in a huge and diverse array of metabolic processes. As such there has been considerable interest over the past two decades towards understanding how Fe-S clusters are formed and distributed to their cognate proteins. A related issue, the topic of the present work, is: why is it that many Fe-S proteins from diverse microbial species cannot be heterologously produced in Escherichia coli in active forms? This issue is of considerable interest not only from the perspective of microbial Fe-S proteins but also for heterologous expression of active eukaryotic Fe-S proteins. The study provides insights on the phylogenetic and biosynthetic limitations concerning formation of functional heterologously expressed Fe-S proteins.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

Reviewed by eLife

Modular, robust and extendible multicellular circuit design in yeast

1. Alberto Carignano
2. Dai Hua Chen
3. Cannon Mallory
4. Clay Wright
5. Georg Seelig
6. Eric Klavins

• Curated by eLife

  Evaluation Summary:

  In this manuscript, the authors address important problems in the field of synthetic biology about scalability, robustness, and modularity. They used multiple strains to build gene circuits and demonstrate the modular composition of strain circuits with an automated design strategy to achieve a target behavior from a large space of possible functional circuit architectures. The major claims of the manuscript are well supported by solid quantitative data and systematic mathematical modeling analysis, and the approaches used are thoughtful and rigorous. This paper is of interest to synthetic biologists within the field of designing community-level behaviors, such as distributed computing, in multicellular consortia.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by eLife

Enhanced Cas12a multi-gene regulation using a CRISPR array separator

1. Jens P. Magnusson
2. Antonio R. Rios
3. Lingling Wu
4. Lei S. Qi

• Curated by eLife

  Evaluation Summary:

  This manuscript is of broad interest to those performing multiplexed genome engineering and related applications with CRISPR Cas12a technologies. While the proposed use of synSeparators is promising, the paper would benefit from further investigation of the mechanism by which synSeparators function to promote Cas12a activity. Additional data would be required to support the current conclusions regarding the generalizability of the findings.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Reviewed by eLife

Mapping the Functional Landscape of the Receptor Binding Domain of T7 Bacteriophage by Deep Mutational Scanning

1. Phil Huss
2. Anthony Meger
3. Megan Leander
4. Kyle Nishikawa
5. Srivatsan Raman

Reviewed by eLife

Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement

1. Astrid Hoermann
2. Sofia Tapanelli
3. Paolo Capriotti
4. Ellen K. G. Masters
5. Tibebu Habtewold
6. George K. Christophides
7. Nikolai Windbichler

Reviewed by eLife