Single-molecule live-cell RNA imaging with CRISPR-Csm.

High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells.

Scarcity of fixed carbon transfer in a model microbial phototroph-heterotroph interaction.

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii's capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing α-proteobacterium.

FIND-seq: high-throughput nucleic acid cytometry for rare single-cell transcriptomics.

Rare cells have an important role in development and disease, and methods for isolating and studying cell subsets are therefore an essential part of biology research. Such methods traditionally rely on labeled antibodies targeted to cell surface proteins, but large public databases and sophisticated computational approaches increasingly define cell subsets on the basis of genomic, epigenomic and transcriptomic sequencing data. Methods for isolating cells on the basis of nucleic acid sequences powerfully complement these approaches by providing experimental access to cell subsets discovered in cell atlases, as well as those that cannot be otherwise isolated, including cells infected with pathogens, with specific DNA mutations or with unique transcriptional or splicing signatures.

Chlamydomonas cells transition through distinct Fe nutrition stages within 48 h of transfer to Fe-free medium.

Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short and long-term adjustments.

The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins.

A protein sequence encodes its energy landscape-all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs-questioning the differences between them and suggesting that both approaches serve as general methods to engineer thermostability.

A map of the rubisco biochemical landscape.

Rubisco is the primary CO fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered where enzyme function is coupled to growth.

Requirements for efficient endosomal escape by designed mini-proteins.

ZF5.3 is a compact, rationally designed mini-protein that escapes efficiently from the endosomes of multiple cell types. Despite its small size (27 amino acids), ZF5.

Tension activation of mechanosensitive two-pore domain K+ channels TRAAK, TREK-1, and TREK-2.

TRAAK, TREK-1, and TREK-2 are mechanosensitive two-pore domain K+ (K2P) channels that contribute to action potential propagation, sensory transduction, and muscle contraction. While structural and functional studies have led to models that explain their mechanosensitivity, we lack a quantitative understanding of channel activation by membrane tension. Here, we define the tension response of mechanosensitive K2Ps using patch-clamp recording and imaging.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay.

Structural and quantum chemical basis for OCP-mediated quenching of phycobilisomes.

Cyanobacteria use large antenna complexes called phycobilisomes (PBSs) for light harvesting. However, intense light triggers non-photochemical quenching, where the orange carotenoid protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells.

Computationally guided AAV engineering for enhanced gene delivery.

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering.

Hachiman is a genome integrity sensor.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman comprises a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease.

Structural basis for the inhibition of PRC2 by active transcription histone posttranslational modifications.

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator essential for embryonic development and maintenance of cell identity that trimethylates histone H3 at lysine 27 (H3K27me3) leading to gene silencing. PRC2 is regulated by association with protein cofactors and crosstalk with histone posttranslational modifications. Trimethylated histone H3 K4 (H3K4me3) and K36 (H3K36me3) localize to sites of active transcription where H3K27me3 is absent and inhibit PRC2 activity through unknown mechanisms.

Stress response silencing by an E3 ligase mutated in neurodegeneration.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress.

Expanding the structural diversity of terpenes by synthetic biology approaches.

Terpenoids display chemical and structural diversities as well as important biological activities. Despite their extreme variability, the range of these structures is limited by the scope of natural products that canonically derive from interconvertible five-carbon (C5) isoprene units. New approaches have recently been developed to expand their structural diversity.

Minimization of the E. coli ribosome, aided and optimized by community science.

The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC).

Spatial profiling of the interplay between cell type- and vision-dependent transcriptomic programs in the visual cortex.

Unlabelled: How early sensory experience during "critical periods" of postnatal life affects the organization of the mammalian neocortex at the resolution of neuronal cell types is poorly understood. We previously reported that the functional and molecular profiles of layer 2/3 (L2/3) cell types in the primary visual cortex (V1) are vision-dependent (Tan et al., (4), 2020; Cheng et al.

A mutation in the low-complexity domain of splicing factor hnRNPA1 linked to amyotrophic lateral sclerosis disrupts distinct neuronal RNA splicing networks.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA-binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in some ALS patients.

Template and target-site recognition by human LINE-1 in retrotransposition.

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA.

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38±10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an assay for RNP formation, and by examining mutant proteins in a viral assembly assay.

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities.

Previous exposure to Spike-providing parental strains confers neutralizing immunity to XBB lineage and other SARS-CoV-2 recombinants in the context of vaccination.

The emergence of SARS-CoV-2 recombinants is of particular concern as they can result in a sudden increase in immune evasion due to antigenic shift. Recent recombinants XBB and XBB.1.