Control Genes with Light

Engineered proteins, tuned to respond to different light levels, control gene expression in bacteria and mammalian cells.

This is from Codon, my weekly newsletter. Subscribe for free.

A new preprint, posted to bioRxiv, reports that photosensitive proteins can be mutated to respond differently to variable levels of light. By introducing these modified proteins into cells, different light intensities can be used to tweak and tune a gene’s expression. Let’s break it down.

A few years ago, in 2017, the same group behind this preprint reported a light-inducible tool to control gene expression, called Opto-T7RNAP. The tool is quite simple. Two “Magnet” proteins, called nMag and pMag, are independently fused to separate halves of the T7 RNA polymerase, a protein that transcribes DNA to RNA.

When pMag and nMag sense light, they attach to one another and bring together the two halves of RNA polymerase. Any genes with a T7 promoter are then ‘switched on.’ This tool offered an easy means to control genes using light, but it was somewhat primitive. Genes either switched ‘on’ or ‘off,’ with few options in-between.

For their latest preprint, the same researchers discovered that if they mutate specific amino acids in nMag and pMag, they can change the light sensitivity of these proteins. Now, a tiny amount of light can drive high levels of gene expression, without causing phototoxicity or heating up the cells.

And “for some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted." That’s a big deal, because now light adds yet another variable for synthetic biologists aiming to create genetic circuits that behave in desired ways.

The variants were identified by randomly mutagenizing specific parts of the proteins, and then screening the mutants in E. coli cells. A total of 14 single, and 5 double, amino acid substitutions were found to change the light sensitivity of the Magnets. Data from several variants are shown below. A fluorescent protein, called mCherry, was used as a test gene for the experiments. Light intensity is plotted along the x-axis, while the levels of mCherry are plotted on the y-axis.

With these tools in hand, one could imagine building a modified Opto-T7RNAP system that can reroute the metabolisms of living cells during fermentation. Lights inside of a bioreactor could switch off to make cells grow rapidly, glow dimly to make cells produce a central metabolite, and then switch on brightly to reprogram cells to convert that metabolite to a final product. Only the future can tell.

Thanks for reading.

— Niko McCarty

Groups of cells, working together, can do things that individuals cannot. This is why microbes, in nature, live in ultra-dense networks. Neighbors share with neighbors. Biologists, by contrast, Extract! Isolate! Silo! If one wants to engineer cells to speak with their neighbors, they’re likely to use small molecules — quorum sensing systems, metabolites, or maybe hormones. This is inherently limited. Little molecules do not encode much information — mainly they are there, or they are not there.

DNA, on the other hand, is a polymer made from four letters that can encode a dazzling array of information. It can store the Guttenberg Bible, or all the instructions to make a little baby. A new preprint uses DNA as an information-rich communication signal between cells.

Specifically, the study uses horizontal gene transfer to send plasmids between cells. By mutating a small piece of these plasmids, one can address the plasmid to specific receiver cells. Each receiver cell encodes a CRISPR system with a unique guide RNA. A received plasmid is only ‘read’ by a cell if its guide RNA does not match the address on the plasmid (see schematic below). And that’s just the beginning. The Caltech researchers behind the preprint also implemented clever strategies to edit DNA messages, pass them along to new recipients, and brilliantly control the flow of information across multiple strains in a population. bioRxiv (Link)

Figure 2 from Marken & Murray. An addressable DNA message is only read by a recipient cell if its own guide RNA does not match the plasmid’s address.

If you want to design a gene therapy to treat a specific, genetic disease, things are conceptually quite simple — just find the problematic gene and devise a strategy to fix the errant nucleotides or replace the gene entirely with a ‘healthy’ copy.

But what do you do when the genetic disease is caused by problems in DNA repair itself? Most CRISPR systems cut DNA, and rely on a cell’s native DNA repair systems to complete the edit. What if a sick cell can’t repair its own DNA?

Fortunately, some of the newer base editor proteins don’t rely on double-stranded DNA breaks. Cleverly, researchers used these new proteins to correct “two of the most prevalent FANCA mutations” — which cause a genetic disorder known as Fanconi Anemia, which affects the bone marrow and causes less blood cells to be made — “in patient hematopoietic stem and progenitor cells.” It’s a gene therapy that makes genetic edits without using DNA repair, in other words.

Initial tests with base editors flopped; editing efficiencies were less than 6% when correcting one type of mutation. Future experiments with the ABE8e base editor (an an evolved enzyme, first reported in 2020 by David Liu’s group), though, were much better: Between 42% and 65% across three groups of patient cells. Nature Communications (Link)


Nearly all of the great tools available to biological engineers — CRISPR, PCR, even next-generation sequencing — are only possible because of basic discoveries. CRISPR comes from microbes, as do the thermostable polymerases that make DNA amplification so simple for a modern biologist. If you want to make the next great discovery, then, it helps to know what is already out there.

A massive database, called SuperNatural 3.0, catalogs nearly 450,000 natural compounds. Thousands of them have anticancer, antibacterial, or other properties. Others are used in makeup and as food preservatives. This database, where possible, also includes “information on pathways, mechanism of action, toxicity,” and even where to buy each chemical. It looks like a great starting point for synthetic biology projects, and the database is freely available online. Nucleic Acids Research (Link)


The Index 🔻

A collection of research papers published in the last week.

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

Basic Research

↑*Enhancing nutritional niche and host defenses by modifying the gut microbiome. Sun Q…Lu TK. Molecular Systems Biology. Link

↑*WiChR, a highly potassium selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells. Vierock J…Hegemann P. Science Advances. Link

  • A new channelrhodopsin for light-mediated control of living cells was discovered in Hyphochytrium catenoides, a soil-dwelling protist. These light-responsive proteins let potassium into cells, rather than the normal sodium, and thus act as an optogenetic inhibitor.

*Hypermutator strains of Pseudomonas aeruginosa reveal novel pathways of resistance to combinations of cephalosporin antibiotics and beta-lactamase inhibitors. Chiang AD…Dekker JP. PLOS Biology. Link

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules. Lim D…Choudhary A. Nature Cell Biology. Link

*A giant virus genome is densely packaged by stable nucleosomes within virions. Bryson TD…Henikoff S. Molecular Cell. Link

PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation. Dhingra Y…Sashital DG. Molecular Cell. Link

Biomanufacturing, Metabolic Engineering & Biomaterials

↑*Conversion of CO2 into organic acids by engineered autotrophic yeast. Baumschabl M…Mattanovich D. PNAS. Link

*Identification of purine biosynthesis as an NADH-sensing pathway to mediate energy stress. Yang R…Li B. Nature Communications. Link

*Ribosome Stalling of N-Linked Glycoproteins in Cell-Free Extracts. Chung SS…DeLisa MP. ACS Synthetic Biology. Link

*Engineered multiple translation initiation sites: a novel tool to enhance protein production in Bacillus licheniformis and other industrially relevant bacteria. Zhang M…Wang Q. Nucleic Acids Research. Link

*Co‑cultivation of anaerobic fungi with Clostridium acetobutylicum bolsters butyrate and butanol production from cellulose and lignocellulose. Brown JL…O’Malley MA. Journal of Industrial Microbiology and Biotechnology. Link

*De novo Synthesis of 2-phenylethanol from Glucose by Metabolically Engineered Escherichia coli. Wang G…Yang X. Journal of Industrial Microbiology and Biotechnology. Link

*Biosynthesis of cannabinoid precursor olivetolic acid in genetically engineered Yarrowia lipolytica. Ma J, Gu Y & Xu P. Communications Biology. Link

Base editing for reprogramming cyanobacterium Synechococcus elongatus. Wang S-Y…Xia P-F. Metabolic Engineering. Link

Improved Production of Fengycin in Bacillus subtilis by Integrated Strain Engineering Strategy. Gao G-R…Yuan Y-J. ACS Synthetic Biology. Link

A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor. Wang R…Nybo SE. ACS Synthetic Biology. Link

The De Novo Synthesis of 2-Phenylethanol from Glucose by the Synthetic Microbial Consortium Composed of Engineered Escherichia coli and Meyerozyma guilliermondii. Yan W…Jiang M. ACS Synthetic Biology. Link

Biosensors

*Pre-equilibrium biosensors as an approach towards rapid and continuous molecular measurements. Maganzini N…Tom Soh H. Nature Communications. Link

Computational Tools & Models

↑*SuperNatural 3.0—a database of natural products and natural product-based derivatives. Gallo K…Banerjee P. Nucleic Acids Research. Link

↑*Molecular de-extinction of ancient antimicrobial peptides enabled by machine learning. Maasch JRMA…de la Fuente-Nunez C. bioRxiv (preprint). Link

*Detection of m6A from direct RNA sequencing using a multiple instance learning framework. Hendra C…Göke J. Nature Methods. Link

*A flexible cross-platform single-cell data processing pipeline. Battenberg K…Minoda A. Nature Communications. Link

*MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters. Terlouw BR…Medema MH. Nucleic Acids Research. Link

*CAMPR4: a database of natural and synthetic antimicrobial peptides. Gawde U…Idicula-Thomas S. Nucleic Acids Research. Link

*Deep mutational scanning and machine learning reveal structural and molecular rules governing allosteric hotspots in homologous proteins. Leander M…Raman S. eLife. Link

*Incorporating Pre-training Paradigm for Antibody Sequence-Structure Co-design. Gao K…Liu T-Y. bioRxiv (preprint). Link

CRISPR, Gene Editing & Control

↑Site-specific CRISPR-based mitochondrial DNA manipulation is limited
by gRNA import. Schmiderer L…Larsson J. Scientific Reports. Link

  • “Since 2015, several papers that claim to successfully adapt
    CRISPR-Cas9 to work on mitochondrial DNA have been published,” says Ludwig Schmiderer. “In this work, we conclusively show that Cas9 does not work in mitochondria because gRNA cannot reach the mitochondrial matrix with current
    technology. We also provide clear guidelines for how future claims of
    mtDNA editing with CRISPR should be validated.”

Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. Chen L…Li D. Nature Biotechnology. Link

↑*A Toolkit for Precise, Multigene Control in Saccharomyces cerevisiae. Sanford A, Kiriakov S & Khalil AS. ACS Synthetic Biology. Link

↑*Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Neugebauer ME…Liu DR. Nature Biotechnology. Link

Expanding the Scope of Bacterial CRISPR Activation with PAM-Flexible dCas9 Variants. Kiattisewee C…Zalatan JG. ACS Synthetic Biology. Link

*High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression. Veggiani G…Sidhu SS. Nature Communications. Link

*CRISPR-based tools for genetic manipulation in pathogenic Sporothrix species. Hatinguais R…da Silva RP. bioRxiv (preprint). Link

DNA Sequencing, Synthesis & Assembly

Metagenome-assembled genome extraction and analysis from microbiomes using KBase. Chivian D…Arkin AP. Nature Protocols. Link

*Mito-SiPE is a sequence-independent and PCR-free mtDNA enrichment method for accurate ultra-deep mitochondrial sequencing. Walsh DJ…Brody LC. Communications Biology. Link

Genetic Circuits

↑*Feedforward growth rate control mitigates gene activation burden. Barajas C…Del Vecchio D. Nature Communications. Link

*Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli. Williams RL & Murray RM. Nature Communications. Link

*Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells. Prochazka L…Zandstra PW. Molecular Systems Biology. Link

A Logically Reversible Double Feynman Gate with Molecular Engineered Bacteria Arranged in an Artificial Neural Network-Type Architecture. Srivastava R & Bagh S. ACS Synthetic Biology. Link

*A programmable reaction-diffusion system for spatiotemporal cell signaling circuit design. Rajasekaran R…Coyle SM. bioRxiv (preprint). Link

Medicine & Diagnostics

↑*An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts. Dousis A…Rabideau AE. Nature Biotechnology. Link

↑*Adenine base editing efficiently restores the function of Fanconi anemia hematopoietic stem and progenitor cells. Siegner SM…Corn JE. Nature Communications. Link

*Safety and immunogenicity of a prefusion non-stabilized spike protein mRNA COVID-19 vaccine: a phase I trial. Gatechompol S…ChulaVAC-001 Study Team. Nature Microbiology. Link

*Safety of the fourth COVID-19 BNT162b2 mRNA (second booster) vaccine: a prospective and retrospective cohort study. Yechezkel M…Yamin D. The Lancet Respiratory Medicine. Link

*Multimeric ACE2-IgM fusions as broadly active antivirals that potently neutralize SARS-CoV-2 variants. Svilenov HL…Buchner J. Communications Biology. Link

*STNM01, the RNA oligonucleotide targeting carbohydrate sulfotransferase 15, as second-line therapy for chemotherapy-refractory patients with unresectable pancreatic cancer: An open label, phase I/IIa trial. Fujisawa T…Yahagi N. eClinicalMedicine. Link

*Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies. Goodman DB…Roybal KT. Science Translational Medicine. Link

CD22-targeted CD28 bispecific antibody enhances antitumor efficacy of odronextamab in refractory diffuse large B cell lymphoma models. Wei J…Skokos D. Science Translational Medicine. Link

*Pore forming–mediated intracellular protein delivery for enhanced cancer immunotherapy. Zhou Z…Sun M. Science Advances. Link

Non-viral precision T cell receptor replacement for personalized cell therapy. Foy SP…Mandl SJ. Nature. Link

*PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries. Henson SN…Ladner JT. Nature Protocols. Link

*Multivalent S2-based vaccines provide broad protection against SARS-CoV-2 variants of concern and pangolin coronaviruses. Halfmann PJ…Kane RS. eBioMedicine. Link

*A click chemistry amplified nanopore assay for ultrasensitive quantification of HIV-1 p24 antigen in clinical samples. Wei X…Liu C. Nature Communications. Link

Plants

THP9 enhances seed protein content and nitrogen-use efficiency in maize. Huang Y…Wu Y. Nature. Link

*Upgrading the genome of an elite japonica rice variety Kongyu 131 for lodging resistance improvement. Wang C…Lin S. Plant Biotechnology Journal. Link

*Enhancing gene editing and gene targeting efficiencies in Arabidopsis thaliana by using an intron-containing version of ttLbCas12a. Schindele P…Puchta H. Plant Biotechnology Journal. Link

Protein & Molecular Engineering

↑*Enhancing the performance of Magnets photosensors through directed evolution. Baumschlager A…Khammash M. bioRxiv (preprint). Link

*Balance-directed protein engineering of IsPETase enhances both PET hydrolysis activity and thermostability. Lee SH…Kim K-J. bioRxiv (preprint). Link

Using enzymes to tame nitrogen-centred radicals for enantioselective hydroamination. Ye Y…Hyster TK. Nature Chemistry. Link

*XNAzymes targeting the SARS-CoV-2 genome inhibit viral infection. Gerber PP…Taylor AI. Nature Communications. Link

*†The Sabatier principle as a tool for discovery and engineering of industrial enzymes. Kari J…Westh P. Current Opinion in Biotechnology. Link

*A bistable and reconfigurable molecular system with encodable bonds. Zhou C…Wang P. Science Advances. Link

*Development of microglia-targeting adeno-associated viral vectors as tools to study microglial behavior in vivo. Okada Y…Hirai H. Communications Biology. Link

*Fragment-based computational design of antibodies targeting structured epitopes. Rangel MA…Sormanni P. Science Advances. Link

*Computationally designed GPCR quaternary structures bias signaling pathway activation. Paradis JS…Barth P. Nature Communications. Link

*Formation of synthetic RNA protein granules using engineered phage-coat-protein -RNA complexes. Granik N…Amit R. Nature Communications. Link

*A glutamine-based single α-helix scaffold to target globular proteins. Escobedo A…Salvatella X. Nature Communications. Link

De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochrome c in cells. Tomasina F…Radi R. PNAS. Link

Tissue Engineering

*Transplantable human thyroid organoids generated from embryonic stem cells to rescue hypothyroidism. Romitti M…Costagliola S. Nature Communications. Link

Tools, Toolkits & Technology

↑*Addressable and adaptable intercellular communication via DNA messaging. Marken JP & Murray RM. bioRxiv (preprint). Link

*Clonal amplification-enhanced gene expression for cell-free directed evolution. Abil Z, Sierra AMR & Danelon C. bioRxiv (preprint). Link

†Modulating cell signalling in vivo with magnetic nanotransducers. Romero G…Anikeeva P. Nature Reviews Methods Primers. Link

*RS-FISH: precise, interactive, fast, and scalable FISH spot detection. Bahry E…Preibisch S. Nature Methods. Link

†Synthetic protein condensates for cellular and metabolic engineering. Qian Z-G, Huang S-C & Xia X-X. Nature Chemical Biology. Link

Programming cell-surface signaling by phase-separation-controlled compartmentalization. Li R…Li P. Nature Chemical Biology. Link

*Ascertaining cells’ synaptic connections and RNA expression simultaneously with barcoded rabies virus libraries. Saunders A…McCarroll SA. Nature Communications. Link

Robotic Construction and Screening of Lanthipeptide Variant Libraries in Escherichia coli. Guo E…Si T. ACS Synthetic Biology. Link

*A simple label-free method reveals bacterial growth dynamics and antibiotic action in real-time. Hammond RJH…Gillespie SH. Scientific Reports. Link

Other Interesting Stuff

↑*Is novel research worth doing? Evidence from peer review at 49 journals. Teplitskiy M…Lakhani KR. PNAS. Link

Epigenetic reversal of hematopoietic stem cell aging in Phf6-knockout mice. Wendorff AA…Ferrando AA. Nature Aging. Link

*Labor advantages drive the greater productivity of faculty at elite universities. Zhang S…Clauset A. Science Advances. Link

*Acquisition of chess knowledge in AlphaZero. McGrath T…Kramnik V. PNAS. Link

*In utero exposure to the Great Depression is reflected in late-life epigenetic aging signatures. Schmitz LL & Duque V. PNAS. Link

A flexible two-photon fiberscope for fast activity imaging and precise optogenetic photostimulation of neurons in freely moving mice. Accanto N…Emiliani V. Neuron. Link