Directed Evolution Tool Doesn't Work: Preprint

A new preprint offers a scathing critique of VEGAS, however, and concludes that it “is not suitable for use as a mammalian directed evolution platform.”

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There were many interesting papers to write about this week. Too many. So today, I’m celebrating a couple preprints that caught my eye. Please also take a look at the papers marked with a ↑ symbol.


Against VEGAS for Directed Evolution

In 2019, a group from UNC-Chapel Hill presented a new method, called VEGAS (viral evolution of genetically actuating sequences), for directed evolution in mammalian cells.

The VEGAS technique uses a Sindbis virus to propagate mutagenized DNA. A gene of interest is first packaged into the virus. That virus is propagated inside of mammalian cells, the gene mutates, and a selection strategy is used to isolate “high fitness variants” of the mutated gene. VEGAS has a reported mutation rate of 10−3 mutations per cycle per base. The technique has been used to “evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks’ time,” according to the initial study.

A new preprint offers a scathing critique of VEGAS, however, and concludes that it “is not suitable for use as a mammalian directed evolution platform.”

Researchers from the University of Sydney and the University of Tasmania, in Australia, found “that the VEGAS system does not result in viral propagation across rounds of replication when performed as described.” The Sindbis virus does package the transgene and does infect cells. But propagation of the virus, after that initial packaging, was unexpectedly halted: viral “cheaters” swiftly emerged and blocked further transgene evolution.

Another little note from this replication study: the VEGAS authors appear to have been unwilling, or unable, to share crucial genetic parts with the Australian team:

“We could not obtain the CMV-SSG plasmid from English et al. and were required to generate this component ourselves … Moreover, English et al. similarly did not provide the original VEGAS circuits required to reproduce their original work, and thus we generated our own serum response factor circuit … We have consulted with English et al. but did not receive advice that resolved these critical technical issues. Together, we conclude that the VEGAS system in its published form is not suitable for directed evolution campaigns.”

Read more at bioRxiv.

Phages + MRI = Detect Bacteria in the Body

Phages are like little Moon Landers. They land on top of bacteria, extrude a “plunger,” and infect cells with their viral genome. Different phages infect different bacteria; many are species-specific. A bacterium, once infected, becomes a factory for new viruses. A bacterium makes this shift towards manufacturing at the cost of its own life.

Now, a new method hijacks phages, using them as a clever tool to image bacteria inside the body using MRI. Here’s how it works:

  1. Find a peptide, or small protein, that binds manganese (the authors found such a peptide in the radiation-proof extremophile, D. radiodurans).
  2. Genetically modify the phage’s ‘coat’ so that it displays this manganese-binding peptide (each phage, in the study, carries about 2,700 copies of this peptide).
  3. Grow some E. coli in an overnight culture. Incubate them with the engineered phage, mix in some manganese, and then image the mixture with MRI (E. coli infected with the phage had a faster relaxation rate, which means that they can be detected with MRI).
  4. In a final experiment, infect V. cholerae with some engineered phage and inject the concoction into the rear ends of mice. Image using MRI; the bacteria show up clearly on the scans.

This paper is obviously a proof-of-concept — they never infected mice with only phages to see if they could infect, say, gut-microbe bacteria — but I’m still a sucker for papers that find new applications for old-school methods.

This technique, notably, was able to detect 1-10 million cells per voxel. So you need quite a lot of bacteria to pick up a signal. The authors think their phages could only day be used for “cell tracking applications involving the use of bacteria as live-cell therapeutics, especially if genetic modification of the bacterial strain to express reporter genes for noninvasive imaging is not desirable or possible.”

We’ll see!

Read more at bioRxiv.

Other Papers

(↑ = recommended article, * = open access, † = review article )

Basic Research

↑↑↑*Directed evolution and selection of biostable L-DNA aptamers with a mirror-image DNA polymerase. Chen J, Chen M & Zhu TF. Nature Biotechnology. Link

↑↑*Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq. Replogle JM…Weissman JS. Cell. Link (Press Release)

*Evolutionary action of mutations reveals antimicrobial resistance genes in Escherichia coli. Marciano DC…Lichtarge O. Nature Communications. Link

*The 3D mutational constraint on amino acid sites in the human proteome. Li B, Roden DM & Capra JA. Nature Communications. Link

*Localization of signaling receptors maximizes cellular information acquisition in spatially structured natural environments. Wang ZJ & Thomson M. Cell Systems. Link


*Cell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria. Vögeli B…Jewett MC. Nature Communications. Link

Efficient production of retinol in Yarrowia lipolytica by increasing stability using antioxidant and detergent extraction. Park H…Hahn J. Metabolic Engineering. Link

Metabolic engineering of the malonyl-CoA pathway to efficiently produce malonate in Saccharomyces cerevisiae. Li S…Deng Y. Metabolic Engineering. Link

*Bioconversion of CO to formate by artificially designed carbon monoxide:formate oxidoreductase in hyperthermophilic archaea. Lim JK…Kim YH. Communications Biology. Link

*Dynamic flux regulation for high-titer anthranilate production by plasmid-free, conditionally-auxotrophic strains of Pseudomonas putida. Fernández-Cabezón L…Nikel PI. Metabolic Engineering. Link

*Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast. Shahsavarani M…Qu Y. bioRxiv (preprint). Link


Repurposing Peptide Nanomaterials as Synthetic Biomolecular Condensates in Bacteria. Tomares DT…Childers WS. ACS Synthetic Biology. Link


*Multimodal detection of dopamine by sniffer cells expressing genetically encoded fluorescent sensors. Herenbrink CK…Herborg F. Communications Biology. Link


*Programmable Mixed-Signal Biocomputers in Mammalian Cells. Letendre JH…Wong WW. bioRxiv (preprint). Link

*A genetic mammalian proportional–integral feedback control circuit for robust and precise gene regulation. Frei T…Khammash M. PNAS. Link

Computational Tools & Models

↑*Machine learning aided construction of the quorum sensing communication network for human gut microbiota. Wu S…Qiao J. Nature Communications. Link

*Design of typical genes for heterologous gene expression. Simm D…Kollmar M. Scientific Reports. Link

*Deep learning driven biosynthetic pathways navigation for natural products with BioNavi-NP. Zheng S…Wu R. Nature Communications. Link

*CRISPRedict: a CRISPR-Cas9 web tool for interpretable efficiency predictions. Konstantakos V…Paliouras G. Nucleic Acids Research. Link

CRISPR & Genetic Control

*Imaging translational control by Argonaute with single-molecule resolution in live cells. Cialek CA…Stasevich TJ. Nature Communications. Link

*Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells. Tao R…Yao S. Nucleic Acids Research. Link

*Compact Cje3Cas9 for Efficient In Vivo Genome Editing and Adenine Base Editing. Chen S…Li Z. The CRISPR Journal. Link

Genome Editing

*Precise CRISPR-Cas–mediated gene repair with minimal off-target and unintended on-target mutations in human hematopoietic stem cells. Tran NT…Chu VT. Science Advances. Link

*HideRNAs protect against CRISPR-Cas9 re-cutting after successful single base-pair gene editing. Harmsen TJW…te Riele H. Scientific Reports. Link

*piggyBac-system-mediated genomic integration of large variant libraries for deep mutational scanning in mammalian cells. Zhao Y…Li S. bioRxiv (preprint). Link

Medicine & Diagnostics

↑*Pre-existing adaptive immunity to the RNA-editing enzyme Cas13d in humans. Tang XE…Yeo GW. Nature Medicine. Link

↑↑↑In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice. Nahmad AD…Barzel A. Nature Biotechnology. Link

↑Potentiating adoptive cell therapy using synthetic IL-9 receptors. Kalbasi A…Garcia KC. Nature. Link

Diverse partial reprogramming strategies restore youthful gene expression and transiently suppress cell identity. Roux AE…Kimmel JC. Cell Systems. Link

Synthetic libraries of immune cells displaying a diverse repertoire of chimaeric antigen receptors as a potent cancer immunotherapy. Fu W…Hu S. Nature Biomedical Engineering. Link

*Neonatal gene therapy achieves sustained disease rescue of maple syrup urine disease in mice. Pontoizeau C…Schiff M. Nature Communications. Link

Targeting C3b/C4b and VEGF with a bispecific fusion protein optimized for neovascular age-related macular degeneration therapy. Yang S…Yu D. Science Translational Medicine. Link

*Re-programming mouse liver-resident invariant natural killer T cells for suppressing hepatic and diabetogenic autoimmunity. Umeshappa CS…Santamaria P. Nature Communications. Link

*†CRISPR-Based Systems for Sensitive and Rapid On-Site COVID-19 Diagnostics. Soh JH…Sabir JSM. Trends in Biotechnology. Link

*Rapid, adaptable and sensitive Cas13-based COVID-19 diagnostics using ADESSO. Casati B…Pecori R. Nature Communications. Link

*Reduction of Pre-Existing Adaptive Immune Responses Against SaCas9 in Humans Using Epitope Mapping and Identification. Shen X…Zhu H. The CRISPR Journal. Link

*Gene Editing of Checkpoint Molecules in Cord Blood-Derived Dendritic Cells and CD8+ T Cells Using CRISPR-Cas9. Presti VL…Nierkens S. The CRISPR Journal. Link


*Point mutations that boost aromatic amino acid production and CO2 assimilation in plants. Yokoyama R…Maeda HA. Science Advances. Link

Protein & Molecular Engineering

↑↑*Designed allosteric protein logic. Plaper T…Jerala R. bioRxiv (preprint). Link

A general approach for engineering RTKs optically controlled with far-red light. Leopold AV…Verkhusha VV. Nature Methods. Link

*Controlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein. Li M…Sun F. Nature Communications. Link

*A hybrid model combining evolutionary probability and machine learning leverages data-driven protein engineering. Illig A…Davari MD. bioRxiv (preprint). Link

Tools & Technology

*Rapid Whole-Genome Identification of High Quality CRISPR Guide RNAs with the Crackling Method. Bradford J, Chappell T & Perrin D. The CRISPR Journal. Link

*Efficient and Fast Generation of Relevant Disease Mouse Models by In Vitro and In Vivo Gene Editing of Zygotes. Sanchez-Baltasar R…García-Bravo M. The CRISPR Journal. Link

*Characterization of ColE1 Production for Robust tolC Plate Dual-Selection in E. coli. Baas-Thomas MS…Church GM. ACS Synthetic Biology. Link

*High-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome. Zheng W…Weitz DA. Science. Link

*BacPROTACs mediate targeted protein degradation in bacteria. Morreale FE…Clausen T. Cell. Link

Simple and Efficient Modification of Golden Gate Design Standards and Parts Using Oligo Stitching. De Saeger J…Jacobs TB. ACS Synthetic Biology. Link


*Using deep learning to detect digitally encoded DNA trigger for Trojan malware in Bio-Cyber attacks. Islam MS…Sri-saan W. Scientific Reports. Link

Discovering Content through Text Mining for a Synthetic Biology Knowledge System. McInnes BT…Nguyen MH. ACS Synthetic Biology. Link

More soon,

— Niko // @NikoMcCarty