Cancer Therapy, Controlled with Sound
Cell Crunch (Issue 2021.03.30)
I have four tips: Curiosity … More curiosity. Even more curiosity. Passion. It’s not enough to be curious.
— Ada Yonath
All of the featured studies this week are preprints, and have not been peer-reviewed.
What’s That Sound?
You’ve probably heard of engineered immune cells being used to treat cancers (think CAR-T), but what about engineered microbes? Some bacteria, it turns out, grow really well near tumors. So why not engineer those bacteria, and coax them into delivering medicines to the nearby cancer cells? That might be a good approach to shrink hard-to-treat tumors.
There’s an obvious problem with this idea; namely, who wants to inject bacteria inside of their body, without knowing where those bacteria are gonna end up? Bacteria circulating through the body can “graft” onto organs and cause damage. We need to find ways to switch on “payload producing” microbes only when they’re near a tumor. And now, a new preprint on bioRxiv, titled, “Acoustic remote control of bacterial immunotherapy,” has done exactly that, showing that engineered bacteria can be engineered to release a drug, while located near a tumor, inside of mice, with sound waves.
How It Works: The team, led by Mikhail Shapiro’s group at Caltech, first identified transcriptional repressors that can be controlled with little heat fluctuations. When these proteins “sense” a sudden shift in temperature, they release their grasp on a gene, and that gene switches on. The researchers found that a specific repressor — called TcI42 — was really good at sensing these temperature shifts. It shut down gene expression strongly at 37 degrees Celsius, but released it’s hold on a gene at 42 degrees Celsius, in response to local heating caused by ultrasound waves. By hooking up that “temperature-sensitive” protein to a gene that encodes a cancer therapy protein, the team could trigger cells to release the therapeutic protein when exposed to ultrasound waves. Cool!
After turning on temperature-controlled genes in bacteria with ultrasound, the team ran into another problem. Cancer therapies have to be delivered over a long time period, usually weeks, and you can’t just blast ultrasound into people for that amount of time (especially if the ultrasound technician gets paid overtime). To fix that issue, Shapiro’s group made a “state switch,” a genetic construct that permanently changes after bacteria are heated up with ultrasound a single time. Blast the microbes with ultrasound, and they’ll continuously produce the therapeutic protein.
In a further batch of experiments, the researchers injected mice with tumor cells, waited for those tumors to grow, and then injected the mice with their ultrasound-controllable cells. After waiting for two days, they focused ultrasound waves into the mice, and then monitored the animals over time. The therapy worked! The bacteria were only activated if they were near the tumors (since that’s where the ultrasound was directed) and they remained active, releasing the therapy, for about two weeks after being switched on. The tumors dwindled in size.
Why It Matters: This isn’t the first time that microbes with therapeutic “payloads” have been used to treat tumors in mice. Tal Danino’s lab, at Columbia University, has been at the forefront of those efforts. But this ultrasound approach will help researchers “activate” only those bacteria located near a tumor, and its entirely non-invasive. Groovy.
Detecting RNA in 20 Minutes
SARS-CoV-2, the virus that causes COVID-19, has an RNA genome. Faster tools to identify specific RNA sequences could help speed up COVID tests. Ideally, a patient could go into a clinic and get a diagnosis in 15 minutes or so. Now, a new medRxiv preprint titled, “Accelerated RNA detection using tandem CRISPR nucleases,” brings that goal within reach.
How It Works: A technique called qRT-PCR is a popular way to test for SARS-CoV-2. It is super sensitive (it can detect just one molecule of RNA per microliter of sample), but also slow and requires technical training.
Instead of using qRT-PCR, a team of researchers — led by Jennifer Doudna’s group at the University of California, Berkeley — turned to CRISPR. They took two CRISPR proteins that, together, can detect RNA and amplify a fluorescent output signal from SARS-CoV-2 in about 20 minutes. The test is sensitive, too — they were able to detect ~30 RNA copies/microliter by combining two proteins: Cas13 and Csm6.
Here’s how they did it: First, they put some Cas13 protein in a tube, and mixed it together with a patient sample and a crRNA, which “guides” the Cas13 protein to an appropriate RNA target (like the SARS-CoV-2 genome). If the crRNA recognizes an RNA sequence within the patient sample, then Cas13 cleaves it and, for lack of a better term, goes ballistic. It begins chopping up nearby RNA sequences, regardless of whether or not the crRNA pairs with them. This is a built-in property of Cas13, and it’s called “collateral activity.”
So Doudna’s lab did a clever thing. They added a modified “Csm6 activator” molecule (called cylic tetra- or hexaadenylates) to the reaction mixture. When these activators bind to a specific part of Csm6, the protein begins to cleave RNA. Now, when Cas13 senses a specific RNA sequence (from SARS-CoV-2, say), its collateral activity switches on and it cuts the modified Csm6 activator molecule. This activates Csm6, which then cleaves a fluorescent reporter molecule, producing a glowing light.
Unfortunately, Csm6 proteins break down the activator molecule over time. After some time, the protein turns off again. This reduces the sensitivity of a diagnostic test. To overcome this barrier, the team chemically modified the “Csm6 activator,” blocking Csm6 from degrading it. That little tweak improved “signal amplification by 100-fold relative to an unmodified activator,” according to the study.
Next, Doudna’s team optimized the proteins and relevant RNA sequences, and tested their “dual protein” method on patient nasopharyngeal swabs. It worked well, returning rapid results even on patient samples that had very few copies of the virus. And by using eight crRNA sequences at once, they could detect “a wide range of SARS-CoV-2 variants.”
Why It Matters: This is not the first time that Cas13 and Csm6 have been used together to detect RNA molecules. Feng Zhang’s group did the same thing in a 2018 Science paper. But that prior study had a low sensitivity (about 1 micromolar of target RNA). The major advancement in this study seems to be the chemical modification of the Csm6 activators, which increased sensitivity about 100-fold. By using multiple crRNAs at once, I suspect you could diagnose just about any SARS-CoV-2 variant.
Building DNA for Fast-Growing Microbes
Vibrio natriegens, a bacterium first isolated in mud, is the “world’s fastest growing organism,” according to a new preprint on bioRxiv, with a doubling time of 10 minutes. Some researchers think it could become the go to microbe for biologists, since experiments could presumably be done in less than half the time of E. coli.
Unfortunately, there hasn’t been an easy-to-use “kit” for engineering V. natriegens, and so it takes a long time to genetically manipulate this microbe. Until now.
Researchers from Georg Fritz’s group at the University of Western Australia have created a Golden Gate library — think LEGO®, but for DNA — specifically for V. natriegens. Their preprint, on bioRxiv, is titled, “The Marburg Collection: A Golden Gate DNA assembly framework for synthetic biology applications in Vibrio natriegens.”
How It Works: This paper presents a “modular cloning” approach for Vibrio natriegens, and it works like this: Each genetic “part” that is typically used to engineer cells — promoters, coding sequences, terminators, and so forth — is stored on its own plasmid. Those “parts” can then be combined into larger transcriptional units, consisting of a promoter, ribosome binding site, coding sequence, and terminator, by using Golden Gate cloning, a technique that leverages restriction enzymes to cut DNA, leave behind “sticky overhangs,” and join together pieces of DNA.
After making transcriptional units, each unit can be combined, again, with other transcriptional units, thus producing plasmids that encode multiple genes.
In this work, the authors create and test dozens of individual components, too (see page 24 of the bioRxiv PDF). There’s 8 different promoters, 11 ribosome binding sites, 10 coding sequences, 12 terminators, and a slew of connectors, antibiotic resistance markers, and other useful ingredients for cooking up your own DNA.
Why It Matters: Vibrio natriegens is a fast-growing microbe, but has not been widely adopted in research labs. This genetic toolkit will help researchers to more rapidly engineer this organism.
🧫 Other Studies Published This Week
- Light-powered reactivation of flagella and contraction of microtubule networks: Toward building an artificial cell. ACS Synthetic Biology. Open Access. Link
- Enabling high‐throughput biology with flexible open‐source automation. Molecular Systems Biology. Open Access. Link
- Cellulose-based biogenic supports, remarkably friendly biomaterials for proteins and biomolecules. Biosensors and Bioelectronics. Link
- Membrane augmented cell-free systems: A new frontier in biotechnology (Review). ACS Synthetic Biology. Link
- Anaerobic conditioning of E. coli cell lysate for enhanced in vitro protein synthesis. ACS Synthetic Biology. Link
- Dual modes of CRISPR-associated transposon homing. Cell. Link
- Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease. Nucleic Acids Research. Open Access. Link
- A class of simple biomolecular antithetic proportional-integral-derivative controllers. bioRxiv (preprint). Link
- Robust and flexible platform for directed evolution of yeast genetic switches. Nature Communications. Open Access. Link
- A rationally engineered decoder of transient intracellular signals. Nature Communications. Open Access. Link
Genetic Engineering & Control
- MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo. Nucleic Acids Research. Open Access. Link
- Simultaneous knock‐out of multiple LHCF genes using single sgRNAs and engineering of a high fidelity Cas9 for precise genome editing in marine algae. Plant Biotechnology Journal. Open Access. Link
- A biologically stable DNAzyme that efficiently silences gene expression in cells. Nature Chemistry. Link
Medicine and Diagnostics
- Development of an engineered probiotic for the treatment of branched chain amino acid related metabolic diseases. Research Square (preprint). Link
- Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis. Cell. Link
- Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection. Nature Biotechnology. Open Access. Link
- Low-cost drug discovery with engineered E. coli reveals an anti-mycobacterial activity of benazepril. bioRxiv (preprint). Link
- A homogeneous split-luciferase assay for rapid and sensitive detection of anti-SARS CoV-2 antibodies. Nature Communications. Open Access. Link
- Characterization of the robust humoral immune response to GSK2618960, a humanized anti-IL-7 receptor monoclonal antibody, observed in healthy subjects in a Phase 1 study. PLOS ONE. Open Access. Link
- Animal immunization, in vitro display technologies, and machine learning for antibody discovery (Review). Trends in Biotechnology. Open Access. Link
- Encapsulin nanocontainers as versatile scaffolds for the development of artificial metabolons. ACS Synthetic Biology. Link
- Highly efficient production of menaquinone-7 from glucose by metabolically engineered Escherichia coli. ACS Synthetic Biology. Link
- Improved architectures for flexible DNA production using retrons across kingdoms of life. bioRxiv (preprint). Link
- Efflux transporters’ engineering and their application in microbial production of heterologous metabolites. ACS Synthetic Biology. Link
- Integrating high cell density cultures with adapted laboratory evolution for improved Gag‐HA virus‐like particles production in stable insect cell lines. Biotechnology and Bioengineering. Link
- Engineered regulon to enable autonomous azide ion biosensing, recombinant protein production, and in vivo glycoengineering. ACS Synthetic Biology. Link
- Isobutanol production by autotrophic acetogenic bacteria. Frontiers in Bioengineering and Biotechnology. Link
- Prospecting biochemical pathways to implement microbe-based production of the new-to-nature platform chemical levulinic acid. ACS Synthetic Biology. Link
- Rational promoter engineering enables robust terpene production in microalgae. ACS Synthetic Biology. Link
- Lineage tracing and analog recording in mammalian cells by single-site DNA writing. Nature Chemical Biology. Link
- A modular tool to query and inducibly disrupt biomolecular condensates. Nature Communications. Open Access. Link
- High-efficiency prime editing with optimized, paired pegRNAs in plants. Nature Biotechnology. Link
- CRISPR/Cas9‐based discovery of maize transcription factors regulating male sterility and their functional conservation in plants. Plant Biotechnology Journal. Open Access. Link
- Application of an integrated computational antibody engineering platform to design SARS-CoV-2 neutralizers. bioRxiv (preprint). Link
Systems Biology & Modelling
- SIGNAL: A web-based iterative analysis platform integrating pathway and network approaches optimizes hit selection from genome-scale assays. Cell Systems. Link
- Biosafety in DIY‐bio laboratories: from hype to policy (Letter). EMBO Reports. Link
- Democratizing biotechnology requires more than availability (Correspondence). Nature Biotechnology. Link
- Refactoring the conjugation machinery of promiscuous plasmid RP4 into a device for conversion of gram-negative isolates to Hfr strains. ACS Synthetic Biology. Link
Until next time,