Unleashed Sense Codons Expand the Ribosome's Potential

A new strain of E. coli can incorporate up to multiple different noncanonical amino acids into its proteins at once. It marks another milestone in a decade-plus long effort.

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☀️ Good morning.

Diseases desperate grown

By desperate appliance are relieved,

Or not at all.

—Shakespeare (Hamlet, Act IV, Scene III)

Unleashing Sense Codons Expands Polymer Powers

A new strain of E. coli can incorporate up to multiple different noncanonical amino acids into its proteins at once. It marks another milestone in a decade-plus long effort from Jason Chin’s group, at the MRC Laboratory of Molecular Biology, in Cambridge, to extend the capabilities of ribosomes for template-directed polymer synthesis.

Though I’m writing about this paper here (obviously), I also suggest reading some terrific technical coverage on this study by Delilah Jewel and Abhishek Chatterjee, as well as a more general news article by Robert Service.

How It Works: For their new study in Science, the Cambridge team began with an E. coli strain that they first reported several years ago. Called Syn61, that strain carries a synthetic, recoded genome. Every single instance of two serine codons (TCG and TCA), as well as every instance of a stop codon, TAG, had been replaced with other codons that encode the same things. The genetic code has three stop codons, and six serine codons. Syn61, then, has 61 codons instead of the typical 64.

To create that Syn61 strain, Chin’s group previously developed a method called REXER (replicon excision for enhanced genome engineering through programmed recombination). Though technically nuanced, REXER basically involves using CRISPR/Cas9 to cut at two places in a genome to excise a large ‘chunk.’ Then, recombination is used to swap in a chemically-synthesized, modified version of that genome chunk. By using REXER again and again, each chunk of the genome can gradually be swapped out with a synthetic, recoded version.

For this latest paper, the researchers continued their work on Syn61; they deleted the cognate tRNAs (serT and serU) for the serine codons, and also deleted release factor 1, a protein that stops translation and liberates new peptides from the ribosome. The new strain, called Syn61.Δ3, is not able to read the normal genetic code. It is thus impervious to a slew of viruses.

Laboratory evolution of the strain turned it from a sluggish Frankenstein’s monster into a fast(er) grower. And by adding trysoyl and pyrrolysyl pairs (a tRNA synthetase and its cognate tRNA) to Syn61.Δ3, the researchers could build proteins that contained two different noncanonical amino acids at up to three different sites in a single protein.

Why It Matters: By expanding the genetic code, and creating polymers with multiple non-canonical amino acids, Chin and his team have opened up a door to creating new protein-based medicines or biomaterials that have user-defined properties.

Credit: Massive Science | Giphy

Base Editing Rescues Sickle Cell Disease in Mice, Patient Cells

Sickle-cell disease is caused by a mutation in a gene called HBB (Hemoglobin subunit beta), which encodes a protein called adult β-globin. Sickle-cell disease affects an estimated 112 people per 100,000 live births globally, and an estimated 1125 people per 100,000 live births in Africa, according to a 2018 study. The disease is characterized by anemia, painful joints, dizziness and vision problems.

For a new study in Nature, researchers at the Broad Institute of Harvard and MIT used an adenine base editor to correct a mutation in HBB, and convert it to a non-pathogenic version. They did this both in haematopoietic stem and progenitor cells from patients that have sickle-cell disease, and in a humanized sickle-cell disease mouse model.

How It Works: The adenine base editor used in this study can convert A•T base pairs to G•C, without causing a double strand break in the DNA, and without donor DNA templates. David Liu’s group first reported this adenine base editor in a 2017 Nature paper. To make it, they fused an evolved Cas9 protein to an evolved transfer RNA adenosine deaminase protein (which can edit RNA via post-transcriptional modifications to mRNA transcripts).

The mutation that caused sickle-cell disease, in this paper, was a single mutation in HBB; an ‘A’ had been changed to a ‘T.’ Liu’s team was able to change that T to a C for this paper. Even though ‘C’ is not the ‘wildtype’ base for the HBB gene, it is a naturally-occurring variant, called ‘hemoglobin Makassar,’ that does not cause the disease.

The team edited haematopoietic stem and progenitor cells from a mouse model of sickle-cell disease, and engrafted them back into mice. The base-edited cells were converted to the Makassar variant at a frequency of 68%, 16 weeks post-transplantation. The edited cells “restored haematological parameters to near-normal levels.”

The team also edited patient CD34+ haematopoietic stem and progenitor cells, again with very high efficiency and low genome-wide off-target effects. When they transplanted those edited human cells into mice, about 70% of the bone marrow in the mice were made up of the human cells after 16 weeks. Their data showed that base-edited human stem cells, taken from people with sickle-cell disease, “can repopulate the haematopoietic system and generate erythroid cells with a greatly reduced propensity for hypoxic sickling.”

Why It Matters: Base editing could be a one-time genetic fix for sickle-cell disease. This study is significant, and takes researchers one step closer towards widespread, clinical relevance for this genetic engineering method. Currently, a bone marrow transplant is the go-to treatment for people with sickle-cell disease.

A Nearly Complete Human Genome

Last week, researchers reported a more complete human genome sequence. The standard reference sequence, in use since about 2013, was missing about 8% of the genome. For a new preprint, though, researchers say that they’ve finished sequencing the human genome, a feat that has been open-ended since the conclusion of the Human Genome Project about two decades ago.

The research was performed by a group called the Telomere-to-Telomere (T2T) Consortium. For this paper, the researchers did not sequence DNA from a living, adult human; rather, they sequenced DNA from a human tissue, called a complete hydatidiform mole, that is made when sperm inseminates a nucleus-free egg. In this study, the sperm carried an X chromosome, and so the researchers did not sequence the Y chromosome.

DNA was sequenced using technology from Pacific Biosciences, in which DNA up to about 20,000 bases in length can be read in a single read. The researchers say that about 0.3% of the new human genome sequence was difficult to “fact-check,” and so there are still some kinks to sort out.

The new work adds about 200 million bases to the former reference genome, and also adds about 115 protein-coding genes.

Next up? The team plans to sequence 300 human genomes in a collaboration with the Human Pangenome Reference Consortium, according to reporting by Nature. They will use their first “complete” human genome sequence as a reference to better understand genetic differences across individuals.

Why It Matters: I mean, what do you want me to say? It’s a more complete human genome sequence. It means more protein-coding genes to study, a better reference for drug trials, and many other benefits for geneticists.

An ABI PRISM 877 integrated thermal cycler, one of the machines used during the Human Genome Project. Credit: National Human Genome Research Institute

🧫 Other Studies This Week


  • Establishment of a Biosensor-based High-Throughput Screening Platform for Tryptophan Overproduction. ACS Synthetic Biology. Link

Cell-Free Systems

  • Longer DNA exhibits greater potential for cell-free gene expression. Scientific Reports (Open Access). Link
  • (Preprint) Decentralizing cell-free RNA sensing with the use of low-cost cell extracts. bioRxiv (Open Access). Link

Directed Evolution

  • An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes. Scientific Reports (Open Access). Link

Fundamental Discoveries

  • (Preprint) A meta-analysis of gRNA library screens enables an improved understanding of the impact of gRNA folding and structural stability on CRISPR-Cas9 activity. bioRxiv (Open Access). Link
  • Production and Characterization of Motile and Chemotactic Bacterial Minicells. ACS Synthetic Biology (Open Access). Link
  • Engineering Gac/Rsm Signaling Cascade for Optogenetic Induction of the Pathogenicity Switch in Pseudomonas aeruginosa. ACS Synthetic Biology. Link
  • CRISPR/Cas9-mediated whole genomic wide knockout screening identifies specific genes associated with PM2.5-induced mineral absorption in liver toxicity. Frontiers in Bioengineering and Biotechnology. Link

Genetic Circuits

  • Cell-Free Characterization of Coherent Feed-Forward Loop-Based Synthetic Genetic Circuits. ACS Synthetic Biology (Open Access). Link

Genetic Engineering & Control

  • Genome editing in mammalian cells using the CRISPR type I-D nuclease. Nucleic Acids Research (Open Access). Link
  • (Preprint) Reprogrammed tracrRNAs enable repurposing RNAs as crRNAs and detecting RNAs. bioRxiv (Open Access). Link

Medicine and Diagnostics

  • Metabolically engineered stem cell–derived exosomes to regulate macrophage heterogeneity in rheumatoid arthritis. Science Advances (Open Access). Link
  • Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell Reports (Open Access). Link
  • Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre. Nature Communications (Open Access). Link
  • (Review) Bioengineering Technologies for Cardiac Regenerative Medicine. Frontiers in Bioengineering and Biotechnology (Open Access). Link

Metabolic Engineering

  • Heterologous biosynthesis of lutein in S. cerevisiae enabled by temporospatial pathway control. Metabolic Engineering (Open Access). Link
  • Engineering the precursor pool to modulate the production of pamamycins in the heterologous host S. albus J1074. Metabolic Engineering (Open Access). Link
  • Dual Regulation of Cytoplasm and Peroxisomes for Improved Α-Farnesene Production in Recombinant Pichia pastoris. ACS Synthetic Biology. Link
  • Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System. ACS Synthetic Biology (Open Access). Link
  • (Review) Molecular characterization of HEK293 cells as emerging versatile cell factories. Current Opinion in Biotechnology (Open Access). Link

Microbial Communities

  • Design of synthetic human gut microbiome assembly and butyrate production. Nature Communications (Open Access). Link
  • (Optochemical) Control of Synthetic Microbial Coculture Interactions on a Microcolony Level. ACS Synthetic Biology. Link

Protein Engineering

  • Genetically Encoding Ultrastable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria. ACS Synthetic Biology. Link
  • (Preprint) Sub-Picomolar Detection of SARS-CoV-2 RBD via Computationally-Optimized Peptide Beacons. bioRxiv (Open Access). Link

Systems Biology, Modelling & Quantitative Studies

  • (Preprint) Distillation of MSA Embeddings to Folded Protein Structures with Graph Transformers. bioRxiv (Open Access). Link
  • (Preprint) Heavy-tailed distributions in a stochastic gene autoregulation model. bioRxiv (Open Access). Link

Tools & Technology

  • Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells. Nature Biotechnology. Link
  • (Preprint) High-throughput discovery of peptide activators of a bacterial sensor kinase. bioRxiv (Open Access). Link
  • Non-invasive and high-throughput interrogation of exon-specific isoform expression. Nature Cell Biology (Open Access). Link
  • A cleavage-based surrogate reporter for the evaluation of CRISPR–Cas9 cleavage efficiency. Nucleic Acids Research (Open Access). Link
  • pegIT - a web-based design tool for prime editing. Nucleic Acids Research (Open Access). Link
  • Establishment of a fluorescent reporter of RNA-polymerase II activity to identify dormant cells. Nature Communications (Open Access). Link
  • (Preprint) NLoed: A Python package for nonlinear optimal experimental design in systems biology. bioRxiv (Open Access). Link
  • noisyR: enhancing biological signal in sequencing datasets by characterizing random technical noise. Nucleic Acids Research (Open Access). Link

Miscellaneous Topics

  • Engineered reproductively isolated species drive reversible population replacement. Nature Communications (Open Access). Link
  • (Review) Protein nanofibrils for next generation sustainable water purification. Nature Communications (Open Access). Link
  • The total number and mass of SARS-CoV-2 virions. PNAS (Open Access). Link

Around the Web

Miscellaneous news.

LEGAL THREATS: A lawyer for Didier Raoult has accused Elisabeth Bik of harassment and blackmail, prompting thousands of academics to pen letters of support for the image sleuth. Science. Link

BIG MONEY: With the economy rebounding, biotech investments have surged, especially in Europe. Labiotech.eu. Link

BACTERIAL TUBES: Bacteria transfer materials, like amino acids and other building blocks, through nanotubes. But it has been challenging to reproduce these structures in the lab. Check out this beautiful, in-depth article by Sruthi Balakrishnan. The Scientist. Link

Until next time,