NEWSLETTER

Protein Barrels by Design & Prime Editing Made Easy

Cell Crunch (Issue 2021.02.22)

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Credit: Doze Studios | Giphy

Build-a-Barrel: Nanopores are tiny holes, nanometers in size, through which molecules can pass. These pores can be created with proteins (which is how many cells receive or excrete nutrients) or with synthetic materials, like silicon and graphene. For a new study, in Science, researchers computationally designed, and created, eight-stranded transmembrane β-barrel proteins that have no homology to known transmembrane β-barrel proteins in nature. The designed proteins readily inserted into synthetic lipid membranes.

In this study, the researchers determined the molecular constraints that membrane embedding imparts on the β-barrel geometries and sequence. Once they settled on a design — after lots of computer simulations — they expressed the de novo proteins in E. coli cells. But, alas, no β-barrels were expressed, likely because these proteins require chaperones to help them fold properly and because the designed sequences had too much positive charge, which impairs protein translation. Eventually, the researchers found a protein sequence, called TMB2.17, that folded fully in vivo and could integrate into the lipid bilayer. They solved the structure of this designed protein at a 2.05-Å resolution. TMB2.17 was very similar, structurally, to the original, computational design. This work was led by the Baker lab at the University of Washington’s Institute for Protein Design.

Why It Matters: Nanopores are useful for biotechnologists and can be used to sequence DNA, for example. Now, designer protein barrels could also pave the way for nanopore-based protein sequencing, which has been much more challenging to develop, as proteins are bulky and there are twenty amino acids in proteins, compared to just four nucleotides in DNA. Designer pores could also be implanted into microbes and used to tune their metabolism, perhaps by filtering out some nutrients, or selectively intaking others. Pores lie at the heart of all cellular metabolism; they are the crucial gatekeepers by which life brings in food and disposes of waste. Now, scientists can design these gates from scratch.

Prime Guide Design: In 2019, David Liu’s group at Harvard University presented a “search-and-replace” genome editing system, called prime editing. Unlike CRISPR/Cas9, which cleaves DNA like a guillotine, prime editing can insert, delete, or introduce point mutations at specific DNA target sites without double-strand breaks. A “prime editor” is made from a Cas9 protein — carrying a H840A mutation — fused to reverse transcriptase. This genome editing technology requires a specific type of guide RNA, called a prime editing guideRNA, to do its duty. The Cas9 nicks one side of the DNA, while the reverse transcriptase enzyme copies a portion of the pegRNA sequence to replace the nicked DNA.

For a new study, published in Nature Communications, researchers in Boston developed PrimeDesign, a web application and command line tool to easily design pegRNAs. They also created a database, called PrimeVar, “that includes candidate prime editing guide RNA (pegRNA) and nicking sgRNA (ngRNA) combinations for installing or correcting >68,500 pathogenic human genetic variants from the ClinVar database.” It sounds like quite a useful resource.

Why It Matters: Prime editing is growing more popular (I suspect) amongst research laboratories. Manually designing pegRNAs can be a frustrating, imprecise effort. A resource that consolidates information is almost always useful for researchers, and can help them save time.

Protein Destructor: Metabolic engineering is all about shifting, deleting and rearranging genes to modify how chemicals are built in living cells. It’s a molecular choose-your-own adventure, with a few rules. Rule #1: Don’t mess with essential genes. Until now.

For a new study, in Nature Communications, researchers used an auxin-inducible protein degradation system to redirect metabolic pathways in yeast. It works like this: auxin is a plant hormone that binds to a specific receptor called, appropriately, the plant auxin receptor. When that happens, a “protein destroyer” complex is summoned in the cell, which chews up the auxin-receptor pair. This natural system has been co-opted by engineers to degrade proteins in other types of cells; not just plants. By attaching a piece of the auxin receptor to a protein in yeast, for example, you can chemically control when a protein is degraded; just add auxin! Unfortunately, sometimes proteins with the auxin receptor bit attached are degraded even when auxin isn’t around. In this study, the researchers — from the Vickers lab at the University of Queensland in Brisbane, Australia — modified the system so that, without auxin present, very little protein degradation takes place.

Why It Matters: The authors applied this auxin-inducible system for three metabolic engineering test cases. In one example, they used the technology to controllably degrade acetyl-CoA carboxylase, an essential gene, in a terpene-producing yeast strain. When they added a bit of auxin to the cells, the cells stopped growing but continued producing a compound called nerolidol. In other words, auxin was used to decouple growth from production, shifting more cellular resources to the latter.

Credit: Hiroshi Nishimasu, F. Ann Ran, Patrick D. Hsu, Silvana Konermann, Soraya I. Shehata, Naoshi Dohmae, Ryuichiro Ishitani, Feng Zhang, and Osamu Nureki | Wikimedia.

Two more studies caught my eye this week. Check out these Twitter threads. 👇

🧫 Other Studies Published This Week

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Biomaterials

• Oxygen-releasing biomaterials: Current challenges and future applications (Review). Trends in Biotechnology. Link

Biosafety

• Advances in synthetic biology, biosafety governance and pandemic control strategies (Review). Frontiers in Bioengineering and Biotechnology. Link

Biosensors

• Bioengineering of genetically encoded gene promoter repressed by flavonoids for constructing intracellular sensor for molecular events. bioRxiv (preprint). Link
• A de novo strategy to develop NIR precipitating fluorochrome for long-term in situ cell membrane bioimaging. PNAS. Link

Fundamental Discoveries

• Clinically relevant mutations in core metabolic genes confer antibiotic resistance. Science. Link
• Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement. Science Advances. Open Access. Link
• Emergence of diauxie as an optimal growth strategy under resource allocation constraints in cellular metabolism. PNAS. Open Access. Link
• Integrative proteomics identifies thousands of distinct, multi-epitope, and high-affinity nanobodies. Cell Systems. Link
• Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages. Scientific Reports. Open Access. Link
• Identification of efficient prokaryotic cell-penetrating peptides with applications in bacterial biotechnology. Communications Biology. Open Access. Link

Genetic Circuits

• Model-guided design of mammalian genetic programs. Science Advances. Open Access. Link

Genetic Engineering & Control

• Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases. Nature Communications. Open Access. Link
• Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination. Nature Communications. Open Access. Link
• Cre-Controlled CRISPR mutagenesis provides fast and easy conditional gene inactivation in zebrafish. Nature Communications. Open Access. Link
• Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights. bioRxiv (preprint). Link
• Expanding the potential of mammalian genome engineering via targeted DNA integration (Review). ACS Synthetic Biology. Link
• Rapid genome engineering of Pseudomonas assisted by fluorescent markers and tractable curing of plasmids. Bio-protocol. Open Access. Link
• Automated rational strain construction based on high-throughput conjugation. ACS Synthetic Biology. Open Access. Link

Medicine and Diagnostics

• Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody. Science. Open Access. Link
• An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants. Science Advances. Open Access. Link
• Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity. Nature Protocols. Link

Metabolic Engineering

• Re-routing of sugar catabolism provides a better insight into fungal flexibility in using plant biomass-derived monomers as substrates. Frontiers in Bioengineering and Biotechnology. Link
• Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases. Nature Protocols. Link
• Engineering Saccharomyces cerevisiae for isoprenol production. Metabolic Engineering. Link
• Production of β‐carotene in Saccharomyces cerevisiae through altering yeast lipid metabolism. Biotechnology and Bioengineering. Link
• Evolving a new efficient mode of fructose utilization for improved bioproduction in Corynebacterium glutamicum. bioRxiv (preprint). Link
• Enzyme engineering and in vivo testing of a formate-reduction pathway. bioRxiv (preprint). Link
• Recent advances in silent gene cluster activation in Streptomyces (Review). Frontiers in Bioengineering and Biotechnology. Link

New Technology

• Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping. Nature Protocols. Link
• SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells. Science Advances. Open Access. Link
• Inference and analysis of cell-cell communication using CellChat. Nature Communications. Open Access. Link
• Microbial single-cell RNA sequencing by split-pool barcoding. Science. Link
• Quantitative yeast–yeast two hybrid for the discovery and binding affinity estimation of protein–protein interactions. ACS Synthetic Biology. Link

Protein Engineering

• Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity. Science. Link
• Absolute and arbitrary orientation of single-molecule shapes. Science. Link
• Ribosome-mediated incorporation of fluorescent amino acids into peptides in vitro. Chemical Communications. Link
• Self-sufficient class VII cytochromes P450: From full-length structure to synthetic biology applications (Review). Trends in Biotechnology. Link
• De novo sequence redesign of a functional Ras binding domain globally inverted the surface charge distribution and led to extreme thermostability. Biotechnology and Bioengineering. Link
• Engineering the immune adaptor protein STING as a biologic. bioRxiv (preprint). Link
• Engineering nucleosomes for generating diverse chromatin assemblies. Nucleic Acids Research. Open Access. Link

Toolkits

• A set of active promoters with different activity profiles for superexpressing Rhodococcus strain. ACS Synthetic Biology. Link
• A programmable toolkit to dynamically signal cells using peptide strand displacement. bioRxiv (preprint). Link

Miscellaneous Topics

• Million-year-old DNA sheds light on the genomic history of mammoths. Nature. Link
• Optical control of fast and processive engineered myosins in vitro and in living cells. Nature Chemical Biology. Link

Have a great week.

— Niko

Thanks for reading Cell Crunch, part of Bioeconomy.XYZ. If you enjoy this newsletter, please share it with a friend or colleague. Reach me with tips and feedback on Twitter @NikoMcCarty or via email.