"Designer" Bacterial Community Treats Colitis in Mice - 2021.05.31
Plus: Other synthetic biology research and news this week.
☀️ Good morning.
The newsletter has returned after a two month hiatus. I’ll send it out once weekly from now on, which I suspect will improve both the quality and the longevity of these updates. Each week, I’ll continue to highlight new research papers and preprints, with relevant news appended to the end of the newsletter. Thank you for reading.
Vacation is what you take when you can't take what you've been taking any longer.
—The Cowardly Lion from The Wizard of Oz
17 Microbes Treat Colitis in Mice
Inflammatory bowel disease (IBD), including Crohn’s and ulcerative colitis, affect an estimated 3 million US adults, according to the CDC.
Current IBD drugs, like adalimumab, are immune modulators, which means that they tweak the immune system to curb inflammation. While drugs like adalimumab work in some patients, though, they can have serious side effects and do little to address upstream inflammatory mechanisms, like the “leaky mucosal barrier, a pro-inflammatory gut microbiome and immunoregulatory defects” that are the hallmarks of these conditions, according to a new study in Nature Communications.
Now, researchers at the University of North Carolina at Chapel Hill have created a “rationally-designed” microbial consortium, consisting of 17 strains, that reduced colon inflammation in a mouse model. Those 17 strains were later reduced to just 11 strains that could do the same thing.
How It Works: To design the initial, 17-member microbial consortium, comprised entirely from publicly-available strains, the researchers first made a list of all the things that go wrong in a patient with IBD, and deliberately chose strains that could address those issues. They picked microbes with known therapeutic functions pertaining to mucosal homeostasis, for example, as well as other microbes that can break down bile salt or release antimicrobials to control opportunistic pathogens in the gut. The team’s initial design, called GUT-103, was effective.
The researchers took some “healthy human fecal material pretreated with chloroform to enrich for spore-forming bacteria” and stuck that into germ-free mice. They next shoved a tube down each mouse’s throat and introduced the GUT-103 microbial community. They found that “GUT-103 rapidly colonized mice, restored normal function to the inflamed colon, and prevented and reversed established experimental colitis,” or colon inflammation, in the animals.
The team next made a refined, 11-member consortium, called GUT108, based on “proprietary human bacterial strains that strongly engrafted and provided similar redundant protective functions.” The 11-member community also reduced inflammation and slashed the number of opportunistic, pathogenic bacteria in a humanized mouse model of colitis.
Why It Matters: Synlogic has several clinical trials underway for “living medicines,” which are engineered bacterial strains that can treat, among other things, phenylketonuria or hyperoxaluria. Biological therapeutics made from microbial consortia has been a more elusive goal, though. This paper represents an intriguing step towards rationally-designed microbial communities that can ameliorate colon inflammation.
My only issue with this paper: the pie and donut charts in Figure 1 and 3.
Hijacked CRISPR Makes Any RNA a Guide
Type II CRISPR systems, which includes the widely-used CRISPR-Cas9, use two components to recognize and cut a DNA target sequence. Those two components are a transactivating crRNA (tracrRNA) and a CRISPR RNA (crRNA).
The tracrRNA first hybridizes to a ‘repeat’ sequence in a crRNA. That hybridized, RNA molecule is then cleaved by an enzyme called RNase III, thus forming a processed crRNA:tracrRNA duplex. This processed molecule guides the Cas enzyme to cut at a specific site — a DNA sequence matching a portion of the crRNA — in the cell’s genome.
It was long thought that crRNAs were encoded only in a specific region of the genome, called the CRISPR-Cas locus. But for a new study, in Science, researchers at the Helmholtz Institute for RNA-based Infection Research, in Germany, show that crRNAs can actually come from RNAs outside of that locus, too.
The researchers brilliantly exploit this finding to develop a CRISPR-based diagnostic platform, called LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection) that can detect numerous viruses at once in a diagnostic test. LEOPARD can even distinguish between SARS-CoV-2 and its D614G (Asp614→Gly) mutant.
How It Works: The researchers first ran a slew of experiments to confirm that cellular RNAs, derived from genome locations away from the CRISPR-Cas locus, can actually form crRNA:tracrRNA duplexes and guide Cas9 to cut target DNA sequences. They did these experiments in both Campylobacter jejuni and Escherichia coli.
In one experiment, the team overexpressed an mRNA sequence for a gene called dctA in E. coli, along with CjeCas9 and a tracrRNA. Together, the components successfully formed in the cells and then targeted and cleaved a DNA target that matched the dctA RNA sequence.
Next, the team engineered the little portion of the tracrRNA that recognizes and binds to crRNAs so that they could more easily pair with cellular mRNAs and form functional Cas9 guides. Their approach was successful. The researchers engineered tracrRNAs for C. jejuni Cas9, as well as the more common SpyCas9 from Streptococcus pyogenes.
Next, the researchers used cellular RNAs to create the LEOPARD diagnostic platform. It works as follows:
In a test tube, add a tracrRNA for your Cas9 protein of interest, together with some Cas9 protein and linear DNA “targets” for each of the pathogens that you’d like to detect. Then, add your test sample (perhaps some sputum?) and let the cellular RNAs from that test sample bind to the tracrRNA. If a cellular RNA from the test sample matches one of the linear DNA targets, that piece of DNA will be cleaved. Finally, run the mixture in a gel, using electrophoresis, and look for any DNA pieces that have been cut. If a patient had SARS-CoV-2, for example, then that virus’ RNA sequence, present in the test sample, would pair with the tracrRNA and cut its corresponding piece of linear DNA. Thus, one can easily distinguish between a slew of pathogens in a single diagnostic test.
In one experiment, the researchers were able to “detect nine ~150-nt RNA fragments associated with respiratory viruses, including two from SARS-CoV-2 coronavirus (the causative agent of COVID-19), six from other coronaviruses, and one from influenza H1N1.” They showed that each of the DNA targets was only cut by Cas9 if its corresponding RNA was present.
Why It Matters: This test is a simple way to test for multiple viruses at once. It could greatly expand the number of pathogens that can be detected by CRISPR-based diagnostics.
XNA Polymerases for Synthetic Biology
Nature is rich with polymers. DNA, proteins, RNA … all are built by cellular machines and woven into long strings, much like beads on a string. Now, synthetic biology is expanding the types of polymers that can be built by cells. Already, researchers have used engineered polymerase enzymes to create chains of 1′,5′-anhydrohexitol nucleic acid or α-Lthreofuranosyl nucleic acids. These spooky-sounding polymers are referred to as synthetic genetic polymers, or XNAs for short. Unfortunately, XNA polymerases are not as good as the normal RNA polymerase. They tend to have low fidelity and are relatively unstable.
For a new study in ACS Synthetic Biology, researchers at the University of California, Irvine, carried out a battery of biochemical tests to assess the “substrate specificity, thermal stability, reverse transcriptase activity, and fidelity of XNA polymerases that were established to synthesize RNA, 2′-fluoroarabino nucleic acid (FANA), arabino nucleic acid (ANA), hexitol nucleic acid (HNA), threose nucleic acid (TNA), and phosphonomethylthreosyl nucleic acid (PMT).”
Each of these engineered polymerases were created by mutating natural versions of RNA polymerase. But in the process of introducing those mutations, write the researchers, the polymerases acquired some obvious flaws. They found that half of the engineered polymerases had diminished activity when heated to above 90°C, for example, and one of the enzymes (called Bst) had “uncontrolled reverse transcriptase activity on HNA.”
Why It Matters: Characterizing proteins may not seem “sexy,” but it’s ridiculously important. To engineer polymerases that can rapidly build other noncanonical polymers, synthetic biologists must first understand how they work. Biochemical assays, then, are an integral part of the design-build-test-learn pipeline.
🧫 Other Studies This Week
Am I missing coverage on a certain topic? Leave a comment on this post.
(Review) Synthetic Biology as Driver for the Biologization of Materials Sciences. Materials Today Bio (Open Access). Link
Versatile artificial mer operons in Escherichia coli towards whole cell biosensing and adsorption of mercury. PLOS ONE (Open Access). Link
(Review) The evolving art of creating genetic diversity: From directed evolution to synthetic biology. Biotechnology Advances (Open Access). Link
The transcriptional landscape of a rewritten bacterial genome reveals control elements and genome design principles. Nature Communications (Open Access). Link
Yeast cell fate control by temporal redundancy modulation of transcription factor paralogs. Nature Communications (Open Access). Link
Evolutionary engineering reveals amino acid substitutions in Ato2 and Ato3 that allow improved growth of Saccharomyces cerevisiae on lactic acid. FEMS Yeast Research. Link
In vivo genome-wide CRISPR screen reveals breast cancer vulnerabilities and synergistic mTOR/Hippo targeted combination therapy. Nature Communications (Open Access). Link
(Preprint) Orthogonal translation initiation using the non-canonical initiator tRNA(AAC) alters protein sequence and stability in vivo. bioRxiv (Open Access). Link
(Preprint) Protein sensors of bacterial kinase activity reveal antibiotic-dependent kinase activation in single cells. bioRxiv (Open Access). Link
(Preprint) Predicting Composition of Genetic Circuits with Resource Competition: Demand and Sensitivity. bioRxiv (Open Access). Link
Genetic Engineering & Control
Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning. Nature Communications (Open Access). Link
(Preprint) Engineering improved Cas13 effectors for targeted post-transcriptional regulation of gene expression. bioRxiv (Open Access). Link
(Preprint) Expansion of the prime editing modality with Cas9 from Francisella novicida. bioRxiv (Open Access). Link
(Review) Large-scale de novo oligonucleotide synthesis for whole-genome synthesis and data storage: challenges and opportunities. Frontiers in Bioengineering and Biotechnology. Link
Medicine and Diagnostics
CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy. Nature Communications (Open Access). Link
Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency. The New England Journal of Medicine. Link
Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq. Nature Communications (Open Access). Link
Rapid single-molecule detection of COVID-19 and MERS antigens via nanobody-functionalized organic electrochemical transistors. Nature Biomedical Engineering (Open Access). Link
(Perspective) CRISPR diagnostics. Science (Open Access). Link
(Review) CRISPR-Cas13 System as a Promising and Versatile Tool for Cancer Diagnosis, Therapy, and Research. ACS Synthetic Biology. Link
Portable bacterial CRISPR transcriptional activation enables metabolic engineering in Pseudomonas putida. Metabolic Engineering. Link
Improved production of human hemoglobin in yeast by engineering hemoglobin degradation. Metabolic Engineering (Open Access). Link
(Preprint) Machine learning-guided acyl-ACP reductase engineering for improved in vivo fatty alcohol production. bioRxiv (Open Access). Link
(Preprint) Increased biosynthesis of acetyl-CoA in the yeast Saccharomyces cerevisiae by overexpression of a deregulated pantothenate kinase gene. bioRxiv (Open Access). Link
(Preprint) A photo-switchable yeast isocitrate dehydrogenase to control metabolic flux through the citric acid cycle. bioRxiv (Open Access). Link
(Review) Towards rational glyco-engineering in CHO: from data to predictive models. Current Opinion in Biotechnology (Open Access). Link
(Review) Engineering Microbes to Bio-Upcycle Polyethylene Terephthalate. Frontiers in Bioengineering and Biotechnology (Open Access). Link
(Review) Recent advances in tuning the expression and regulation of genes for constructing microbial cell factories. Biotechnology Advances. Link
Synthetic neural-like computing in microbial consortia for pattern recognition. Nature Communications (Open Access). Link
Drawing up a collaborative contract: Amino acid cross-feeding between inter-species bacterial pairs. Biotechnology and Bioengineering. Link
Improved plant cytosine base editors with high editing activity, purity, and specificity. Plant Biotechnology Journal (Open Access). Link
Stepwise artificial evolution of an Sw-5b immune receptor extends its resistance spectrum against resistance-breaking isolates of Tomato spotted wilt virus. Plant Biotechnology Journal (Open Access). Link
(Review) Engineering healthy crops: molecular strategies for enhancing the plant immune system. Current Opinion in Biotechnology. Link
(Preprint) Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells. bioRxiv (Open Access). Link
(Review) Protein sequence design with deep generative models. Current Opinion in Chemical Biology (Open Access). Link
Systems Biology, Modelling & Quantitative Studies
(Preprint) Parameterization of regulatory nodes for engineering broad host range heterologous gene expression. bioRxiv (Open Access). Link
Pareto optimality between growth-rate and lag-time couples metabolic noise to phenotypic heterogeneity in Escherichia coli. Nature Communications (Open Access). Link
Coordination of gene expression noise with cell size: analytical results for agent-based models of growing cell populations. Journal of the Royal Society Interface. Link
Optimization of Electrotransformation Parameters and Engineered Promoters for Lactobacillus plantarum from Wine. ACS Synthetic Biology. Link
Tools & Technology
CRISPECTOR provides accurate estimation of genome editing translocation and off-target activity from comparative NGS data. Nature Communications (Open Access). Link
BAR-Seq clonal tracking of gene-edited cells. Nature Protocols. Link
Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo. Nature Communications (Open Access). Link
(Preprint) A ratiometric dual-color luciferase reporter for fast characterization of transcriptional regulatory elements. bioRxiv (Open Access). Link
A global metagenomic map of urban microbiomes and antimicrobial resistance. Cell (Open Access). Link
(Obituary) Dan Salah Tawfik (1955-2021)—A giant of protein evolution. EMBO Reports (Open Access). Link
(Preprint) Genetically engineered coral: A mixed-methods analysis of initial public opinion. bioRxiv (Open Access). Link
Strengthening the United Nations Secretary-General’s Mechanism to an alleged use of bioweapons through a quality-assured laboratory response. Nature Communications (Open Access). Link
✨ Around the Web
A newsletter section devoted to miscellaneous synthetic biology news.
SYNBIO POX: Synthetic biology can be used to re-create viruses, such as smallpox. Should we be worried? The dek of this WIRED article, at least, makes it seem like a scary prospect. WIRED. Link
OLD MOLE: A naked mole rat named Joe turns 39 years old, the highest age ever recorded for the species. Will Joe help scientists unravel the secrets of longevity? WIRED. Link
LET THERE BE SIGHT: A French research team used optogenetics to restore vision in a patient with retinitis pigmentosa, a genetic disorder that causes gradual deterioration of the retina. Ars Technica. Link (Also covered in MIT Technology Review and The Scientist).
HAMSTER HELP: A DNA vaccine, developed by researchers in Taiwan, protected hamsters from SARS-CoV-2. Fierce Biotech. Link
CRISPR NFTs: UC Berkeley is auctioning off two NFTs, or nonfungible tokens, in the coming weeks. One of those NFTs is the patent disclosure form describing CRISPR-Cas9, penned by Doudna and Charpentier. The New York Times. Link
G.M.O. EASE: The UK is expected to announce an easing to their current regulations for some gene-edited crops and livestock on June 17. Science. Link
HUMAN EMBRYO GUIDE: The International Society for Stem Cell Research has released a new set of guidelines that includes less restrictive policies on lab-grown human embryos. The guidelines previously prohibited human embryo cultures 14 days post-fertilization. Science. Link
Until next time,