helixCAM: A platform for programmable cellular assembly in bacteria and human cells.

Interactions between cells are indispensable for signaling and creating structure. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, endogenous CAMs are natively expressed by many cells and tend to have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase "helixCAM," a platform for engineering synthetic CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. Based on coiled-coil interaction principles, we built a set of rationally designed helixCAM libraries, which led to the discovery of additional high-performance helixCAM pairs. We applied this helixCAM toolkit for various multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning.

A Deep Learning Approach to Antibiotic Discovery.

Due to the rapid emergence of antibiotic-resistant bacteria, there is a growing need to discover new antibiotics. To address this challenge, we trained a deep neural network capable of predicting molecules with antibacterial activity. We performed predictions on multiple chemical libraries and discovered a molecule from the Drug Repurposing Hub-halicin-that is structurally divergent from conventional antibiotics and displays bactericidal activity against a wide phylogenetic spectrum of pathogens including Mycobacterium tuberculosis and carbapenem-resistant Enterobacteriaceae. Halicin also effectively treated Clostridioides difficile and pan-resistant Acinetobacter baumannii infections in murine models. Additionally, from a discrete set of 23 empirically tested predictions from >107 million molecules curated from the ZINC15 database, our model identified eight antibacterial compounds that are structurally distant from known antibiotics. This work highlights the utility of deep learning approaches to expand our antibiotic arsenal through the discovery of structurally distinct antibacterial molecules.

An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance.

Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.

A swapped genetic code prevents viral infections and gene transfer.

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer1-6. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus7-9, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature10. Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.

Recording gene expression order in DNA by CRISPR addition of retron barcodes.

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons1, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.

Realizing the potential of synthetic biology.

Synthetic biology, despite still being in its infancy, is increasingly providing valuable information for applications in the clinic, the biotechnology industry and in basic molecular research. Both its unique potential and the challenges it presents have brought together the expertise of an eclectic group of scientists, from cell biologists to engineers. In this Viewpoint article, five experts discuss their views on the future of synthetic biology, on its main achievements in basic and applied science, and on the bioethical issues that are associated with the design of new biological systems.

Reprogramming to recover youthful epigenetic information and restore vision.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.

Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions.

Antibodies have long served as vital tools in biological and clinical laboratories for the specific detection of proteins. Conventional methods employ fluorophore or horseradish peroxidase-conjugated antibodies to detect signals. More recently, DNA-conjugated antibodies have emerged as a promising technology, capitalizing on the programmability and amplification capabilities of DNA to enable highly multiplexed and ultrasensitive protein detection. However, the nonspecific binding of DNA-conjugated antibodies has impeded the widespread adoption of this approach. Here, we present a novel DNA-conjugated antibody staining protocol that addresses these challenges and demonstrates superior performance in suppressing nonspecific signals compared to previously published protocols. We further extend the utility of DNA-conjugated antibodies for signal-amplified in situ protein imaging through the hybridization chain reaction (HCR) and design a novel HCR DNA pair to expand the HCR hairpin pool from the previously published 5 pairs to 13, allowing for flexible hairpin selection and higher multiplexing. Finally, we demonstrate highly multiplexed in situ protein imaging using these techniques in both cultured cells and tissue sections.