Controversial Study Explains How to Engineer the Coronavirus: Index #22

A new protocol, published in Nature Protocols, describes a reverse genetic system to create SARS-CoV-2 viruses with desired mutations.

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There’s too many studies to cover each week. That’s a good thing. But to include all of them—and avoid making decisions—I’ve switched up the format of this newsletter. I’d appreciate your feedback through this one question survey.

We live in a society absolutely dependent on science and technology and yet have cleverly arranged things so that almost no one understands science and technology. That's a clear prescription for disaster. - Carl Sagan

How to Engineer SARS-CoV-2: A new protocol, published in Nature Protocols, describes a reverse genetic system to create SARS-CoV-2 viruses with desired mutations. Creating the coronavirus—which is about 30,000 nucleotides in length—requires six basic steps, each of which could likely be completed by an undergraduate student in molecular biology. First, plasmids are prepared that complement each part of the virus, then those plasmids are cut and stitched together, before being converted to RNA and inserted into cells, which produce the viruses. The work was authored by researchers at the University of Texas Medical Branch, in Galveston, Texas.

Why It Matters: There are several SARS-CoV-2 variants circulating globally, according to the U.S. Centers for Disease Control and Prevention. To study new variants of the coronavirus—or variants that are likely to emerge—researchers can create them in the lab, and observe how specific mutations alter their properties. This protocol explains how to do that with a basic knowledge of molecular biology, and somewhat commonplace lab equipment, making it a potential biosecurity concern. Synthetic biologists expressed concern about the study on Twitter.

Transmission electron micrograph of SARS-CoV-2 virus particles. [Credit: NIAID | Wikimedia CC BY 2.0]

Microbial Communities: For a new study, published in Nature Communications, researchers used Bayesian statistics to find optimal combinations of bacteria to build microbial communities. The study focuses on two different methods to build microbial communities: quorum sensing (which are cellular signals based on small molecules) and bacteriocins, antimicrobial proteins that kill nearby bacteria and can be used to control their growth. The work was mathematical in nature, and the next step will be to test their predictions in the laboratory. The study was authored by researchers at University College London. To learn more about this study, read this article from the first author, Behzad Karkaria.

Why It Matters: By working together, bacterial organisms can perform tasks that, alone, would be impossible. For example, groups of different bacteria can work together to create complex molecules, or to share the “burden” of a particularly troublesome metabolic pathway. Unfortunately, it is hard to create microbial communities that can live, and work, together for a long time. A single type of bacteria in the mixture can dominate the others, destroying the community. In this study, the researchers “were able to derive the fundamental interactions that are most commonly associated with stable communities,” providing a framework to more easily, and efficiently, create synthetic microbial communities. This will help synthetic biologists expand their work from one organism to several.

More CRISPR Targets: For a new preprint, posted to bioRxiv, researchers showed that the amount of ‘AT’ or ‘GC’ nucleotides in a certain type of CRISPR array affects the performance of a Cas protein, called Cas12a. This study was authored by researchers at Stanford University.

Why It Matters: CRISPR is the gene-editing tool of choice, largely supplanting historical methods for manipulating DNA. The most commonly used protein to cut DNA, today, is probably the Cas9 protein taken from a bacterium called Streptococcus pyogenes. But there are other Cas proteins. One of them, called Cas12a, can even process its own guide RNA arrays, a feature that makes it well-suited to targeting—and cutting—many DNA targets at once. A study from 2019 showed that Cas12a can edit 25 genetic targets at once, and that its guide RNAs can be stored and expressed from a single piece of DNA. This new preprint demonstrates that the GC or AT content of spacer sequences between those guide RNAs affects how well they are expressed and, thus, how well Cas12a will work. This study could make “multiplexed” DNA editing—where many genes are targeted at once—more efficient.

Open-Source Research Software: In a paper published in Synthetic Biology, researchers at the University of Washington, in Seattle, present an open-source software, called Aquarium. The software can be used to manage experiments and laboratory inventory (think chemicals, pipette tips and gloves), and even store protocols and data.

Why It Matters: Many scientists use a hodgepodge of tools to manage their work. During my time in research labs, I used Benchling to take notes and design DNA sequences, Dropbox to store data files and Quartzy to manage lab inventories. Aquarium packs most of these features into one place. Experiments can be planned, in the software, through a graphical user interface, and then presented as a ‘workflow’ where each step, once completed, triggers the next step in the protocol. The software also seems pretty smart—it will warn you if there’s likely to be contamination in an experiment, or if you’re over budget for the month.

TALEN Beats CRISPR: In a new study, published in Nature Communications, researchers at the University of Illinois at Urbana−Champaign showed that TALEN, which stands for transcription activator-like effector nucleases, are more efficient at cutting DNA in tightly packed heterochromatin than the Cas9 protein. A TALEN is a type of protein that is made by fusing a TAL effector protein—which can bind to DNA—to a protein that can cut DNA. These proteins have been used to cut DNA since at least 1996.

Why It Matters: Cas9 is the de facto protein for cutting DNA. This new study, though, presents a better alternative to cut DNA inside of heterochromatin, which makes up an estimated “∼25% to 90% of multicellular eukaryotic genomes,” according to a 2018 review. To figure this out, the researchers used single-molecule imaging, in living cells, and found that Cas9 is not as good at cutting this type of DNA because it “becomes encumbered by local searches on non-specific sites,” unlike TALEN.

DNA condenses. [Credit: College of Natural Sciences, UT Austin | Giphy]

🧫 Other Studies Published This Week


  • Genetically encoded formaldehyde sensors inspired by a protein intra-helical crosslinking reaction. Nature Communications. Open Access. Link
  • A whole-cell biosensor for point-of-care detection of waterborne bacterial pathogens. ACS Synthetic Biology. Open Access. Link

Cell-Free Systems

  • A Streptomyces venezuelae cell-free toolkit for synthetic biology. ACS Synthetic Biology. Open Access. Link

Directed Evolution

  • ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces. bioRxiv. Open Access. Link

Fundamental Discoveries

  • CRISPR/Cas9-mediated genome engineering reveals the contribution of the 26S proteasome to the extremophilic nature of the yeast Debaryomyces hansenii. ACS Synthetic Biology. Link
  • Genome-wide CRISPR-Cas9 screen identified KLF11 as a druggable suppressor for sarcoma cancer stem cells. Science Advances. Open Access. Link
  • A biofoundry workflow for the identification of genetic determinants of microbial growth inhibition. Synthetic Biology. Open Access. Link
  • A novel all-in-one conditional knockout system uncovered an essential role of DDX1 in ribosomal RNA processing. Nucleic Acids Research. Open Access. Link
  • Integrated spatial genomics reveals global architecture of single nuclei. Nature. Link
  • Global analysis of protein arginine methylation. bioRxiv. Open Access. Link

Genetic Circuits

  • A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues. Science Advances. Open Access. Link
  • A synthetic switch based on orange carotenoid protein to control blue light responses in chloroplasts. bioRxiv. Open Access. Link

Genetic Engineering

  • In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing. Nature Communications. Open Access. Link
  • Designing P. aeruginosa synthetic phages with reduced genomes. Scientific Reports. Open Access. Link
  • (Review) Genetic toolkits to design and build mammalian synthetic systems. Trends in Biotechnology. Link
  • Guide-target mismatch effects on dCas9–sgRNA binding activity in living bacterial cells. Nucleic Acids Research. Open Access. Link
  • Genetic code expansion of Vibrio natriegens. Frontiers in Bioengineering and Biotechnology. Link

Medicine and Diagnostics

  • In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA. Nature Biomedical Engineering. Link
  • Direct control of CAR T cells through small molecule-regulated antibodies. Nature Communications. Open Access. Link
  • A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Science Advances. Open Access. Link
  • Gene therapy via canalostomy approach preserves auditory and vestibular functions in a mouse model of Jervell and Lange-Nielsen syndrome type 2. Nature Communications. Open Access. Link

Metabolic Engineering

  • Development of the high-productivity marine microalga, Picochlorum renovo, as a photosynthetic protein secretion platform. Algal Research. Link
  • Engineering lithoheterotrophy in an obligate chemolithoautotrophic Fe(II) oxidizing bacterium. Scientific Reports. Open Access. Link
  • (Review) Metabolic Engineering of Cupriavidus necator H16 for Sustainable Biofuels from CO2. Trends in Biotechnology. Open Access. Link
  • Transportome-wide engineering of Saccharomyces cerevisiae. Metabolic Engineering. Open Access. Link
  • Extending the shikimate pathway for microbial production of maleate from glycerol in engineered E. coli. Biotechnology and Bioengineering. Link
  • Fermentative production of L-2-Hydroxyglutarate by engineered Corynebacterium glutamicum via pathway extension of L-lysine biosynthesis. Frontiers in Bioengineering and Biotechnology. Open Access. Link
  • Efficient production of oxidized terpenoids via engineering fusion proteins of terpene synthase and cytochrome P450. Metabolic Engineering. Link

New Technology

  • Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems. Science. Link
  • A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nature Communications. Link
  • Light-controllable RNA-protein devices for translational regulation of synthetic mRNAs in mammalian cells. Cell Chemical Biology. Link
  • Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope. Nature Protocols. Link
  • The loopometer: a quantitative in vivo assay for DNA-looping proteins. Nucleic Acids Research. Open Access. Link
  • Single cell epigenetic visualization assay. Nucleic Acids Research. Open Access. Link
  • Low-cost, scalable, and automated fluid sampling for fluidics applications. bioRxiv. Open Access. Link

Protein Engineering

  • De novo design of modular and tunable protein biosensors. Nature. Open Access. Link
  • Orthogonal translation enables heterologous ribosome engineering in E. coli. Nature Communications. Open Access. Link
  • DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously. Nature Communications. Open Access. Link
  • Cell-free directed evolution of a protease in microdroplets at ultrahigh throughput. ACS Synthetic Biology. Open Access. Link
  • Pore structure controls stability and molecular flux in engineered protein cages. bioRxiv. Open Access. Link
  • Posttranslational chemical installation of azoles into translated peptides. Nature Communications. Open Access. Link

Systems Biology and Modelling

  • Benchmarking of numerical integration methods for ODE models of biological systems. Scientific Reports. Open Access. Link
  • Dynamical modeling of optogenetic circuits in yeast for metabolic engineering applications. ACS Synthetic Biology. Link
  • Probability-based mechanisms in biological networks with parameter uncertainty. bioRxiv. Open Access. Link
  • Generating novel protein sequences using Gibbs sampling of masked language models. bioRxiv. Open Access. Link

Have a great week.