100,000 Gene Delivery Vehicles: Adeno-associated viruses (AAVs) are equipped with a protein coat, or capsid, which encases the virus's genetic material. This capsid can also incorporate DNA encoding for CRISPR components, allowing AAVs to serve as vehicles for gene therapy. In a recent study published in Nature Biotechnology, researchers engineered 110,689 distinct viable AAV2 capsids by introducing random mutations into a targeted segment of the protein sequence. They developed a deep learning algorithm, a variant of artificial neural networks, which successfully predicted which DNA sequences could yield functional AAV2 capsids. While most AAVs generated were non-viable due to improper assembly or failure to package DNA, about 58% of AAV2s with a single mutation proved viable, whereas only 0.3% of those with six mutations were functional. This research was spearheaded by teams from Google alongside the Wyss Institute at Harvard and MIT.

Why It Matters: By deciphering the design principles of AAV2 vectors—through the creation of over 100,000 variants and the subsequent utilization of this dataset to train a deep learning model—scientists can develop gene therapy approaches that are more specific and less likely to invoke an immune response. Understanding which DNA sequences can effectively form viable capsids is crucial as researchers aim to create larger AAV2 vectors capable of carrying more genetic material for therapies, potentially saving significant time in the research process.

Immune-Resistant Vectors: Further insights into AAVs reveal that they can sometimes provoke an immune response. These viruses, viewed as foreign entities, bind to a receptor known as Toll-like receptor 9 (TLR9), leading to the release of pro-inflammatory cytokines. In a study featured in Science Translational Medicine, scientists from Harvard University engineered AAVs designed to minimize immune activation. By attaching short, single-stranded DNA sequences to AAV capsids, they effectively hindered TLR9's function. These sequences could form "stem-loop structures" that fit into TLR9's ring structure, blocking its dimerization and subsequent activation. Testing in mice, pigs, and macaque monkeys showed promising results with the modified AAVs eliciting no immune response in many scenarios, although inflammation was observed when injected into the vitreous humor of macaque monkeys.

Why It Matters: The increasing prevalence of gene therapies necessitates AAVs that are both safe and effective for targeted tissue delivery. This innovative approach to dampening immune responses triggered by AAVs represents a significant advancement, with potential for further refinement in future research.

Enhanced Artificial Cells: Certain bacteria, such as Rhodobacter sphaeroides, possess chromatophores, which are vesicles that enable photosynthesis. A recent study published in the Proceedings of the National Academy of Sciences explored the incorporation of these chromatophores into larger, artificial cells. The chromatophores were capable of generating energy (in the form of ATP) from ADP, thus energizing the larger cells akin to quasi-mitochondria. The research team utilized cryo-electron microscopy to examine the spatial organization of ATP-producing proteins (ATP synthase), which can generate approximately 100 ATP molecules per second. This work was conducted by researchers at the University of Bari Aldo Moro in Italy.

Why It Matters: Synthetic biologists focused on bottom-up approaches aim to construct living cells from fundamental chemical components. Current artificial cells are often simplistic and have limited energy sources, either being finite or externally supplied. To create more complex cells from the ground up, continuous internal energy production is essential. This study marks a crucial step toward achieving that ambitious goal.

Other Studies Published This Week

Artificial Cells - Reconstituting natural cell elements in synthetic cells (Review). Advanced Biology. [Link](#) - Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells. PNAS. Open Access. [Link](#)

Biosensors - Structure- and mechanism-guided design of single fluorescent protein-based biosensors. Nature Chemical Biology. [Link](#) - A high-performance genetically encoded fluorescent biosensor for imaging physiological peroxynitrite. Cell Chemical Biology. [Link](#) - CRISPRi-mediated NIMPLY logic gate for fine-tuning whole-cell sensing toward simple urine glucose detection. ACS Synthetic Biology. [Link](#) - Macrolide biosensor optimization through cellular substrate sequestration. ACS Synthetic Biology. [Link](#) - A reporter system for cytosolic protein aggregates in yeast. ACS Synthetic Biology. Open Access. [Link](#) - A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics. bioRxiv. Open Access. [Link](#)

Fundamental Discoveries - A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry. Nature Communications. Open Access. [Link](#) - Systematic characterization of mutations altering protein degradation in human cancers. Molecular Cell. [Link](#) - Construction of intracellular asymmetry and asymmetric division in Escherichia coli. Nature Communications. Open Access. [Link](#) - Structural robustness affects the engineerability of aminoacyl-tRNA synthetases for genetic code expansion. Biochemistry. [Link](#) - Intercellular communication induces glycolytic synchronization waves between individually oscillating cells. PNAS. Open Access. [Link](#) - A DNA-origami NanoTrap for studying the diffusion barriers formed by Phe-Gly-rich nucleoporins. bioRxiv. Open Access. [Link](#) - Addressing evolutionary questions with synthetic biology. OSF preprint. Open Access. [Link](#)

Genetic Circuits - Synthetic multistability in mammalian cells. bioRxiv. Open Access. [Link](#) - Winner-takes-all resource competition redirects cascading cell fate transitions. Nature Communications. Open Access. [Link](#) (Read the press release.) - A burden-free gene overexpression system. bioRxiv. Open Access. [Link](#) - Emergent oscillation induced by nutrient-modulating growth feedback. bioRxiv. Open Access. [Link](#)

Genetic Engineering & Control - Engineering 3D genome organization (Review). Nature Reviews Genetics. Open Access. [Link](#) - Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells. Biotechnology and Bioengineering. [Link](#) - Transcriptional control of Clostridium autoethanogenum using CRISPRi. Synthetic Biology. Open Access. [Link](#) - Small antisense DNA-based gene silencing enables cell-free bacteriophage manipulation and genome replication. ACS Synthetic Biology. [Link](#) - Programmable tools for targeted analysis of epigenetic DNA modifications (Review). Current Opinion in Chemical Biology. Open Access. [Link](#) - The yeast platform engineered for synthetic gRNA-landing pads enables multiple gene integrations by a single gRNA/Cas9 system. Metabolic Engineering. [Link](#)

Medicine and Diagnostics - Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape. Science. Open Access. [Link](#) - Human heart-forming organoids recapitulate early heart and foregut development. Nature Biotechnology. [Link](#) - Synthetic biology in the clinic: engineering vaccines, diagnostics, and therapeutics (Review). Cell. [Link](#)

Metabolic Engineering - Optimized gene expression from bacterial chromosome by high-throughput integration and screening. Science Advances. Open Access. [Link](#) - Improving the production of NAD+ via multi-strategy metabolic engineering in Escherichia coli. Metabolic Engineering. [Link](#) - Quorum sensing-mediated protein degradation for dynamic metabolic pathway control in Saccharomyces cerevisiae. Metabolic Engineering. [Link](#) - Metabolic engineering of Saccharomyces cerevisiae for ethyl acetate biosynthesis. ACS Synthetic Biology. [Link](#) - Engineering biocatalytic solar fuel production: The PHOTOFUEL consortium (Review). Trends in Biotechnology. [Link](#) - Engineering Halomonas bluephagenesis as a chassis for bioproduction from starch. Metabolic Engineering. [Link](#) - Successful enzyme colocalization strategies in yeast for increased synthesis of non-native products (Review). Frontiers in Bioengineering and Biotechnology. Open Access. [Link](#) - Lactic acid bacteria-derived ?-glucans: From enzymatic synthesis to miscellaneous applications (Review). Biotechnology Advances. [Link](#)

New Technology - Programmable human histone phosphorylation and gene activation using a CRISPR/Cas9-based chromatin kinase. Nature Communications. Open Access. [Link](#) - Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics. Nature Biotechnology. [Link](#) - RNA secondary structure prediction using deep learning with thermodynamic integration. Nature Communications. Open Access. [Link](#) - Optimized CRISPR tools and site-directed transgenesis in Culex quinquefasciatus mosquitoes for gene drive development. bioRxiv. Open Access. [Link](#) - cytoNet: Spatiotemporal network analysis of cell communities. bioRxiv. Open Access. [Link](#)

Plants - Towards plant resistance to viruses using protein-only RNase P. Nature Communications. Open Access. [Link](#) - Genome engineering for crop improvement and future agriculture (Review). Cell. [Link](#)

Protein Engineering - Self-assembly and regulation of protein cages from pre-organised coiled-coil modules. Nature Communications. Open Access. [Link](#) - Complete and cooperative in vitro assembly of computationally designed self-assembling protein nanomaterials. Nature Communications. Open Access. [Link](#) - Designed folding pathway of modular coiled-coil-based proteins. Nature Communications. Open Access. [Link](#)

Systems Biology and Modelling - Model reduction of genome-scale metabolic models as a basis for targeted kinetic models. Metabolic Engineering. Open Access. [Link](#)

Wishing you a productive week ahead.

— Niko

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