Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria.

Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.

Programmable synthetic biology tools for developing microbial cell factories.

Microbial conversion to generate value-added chemicals from diverse biomass is one of the keystones of energy biotechnology. Programmable synthetic biology tools offer versatile, standardized options for developing microbial cell factories. These tools thus can be reprogrammed in a user-defined manner for flexible wiring of stimuli and response, highly efficient genome engineering, and extensive perturbation of metabolic flux and genetic circuits. They also can be modularly assembled to construct elaborate and unprecedented biological systems with unique features. This review highlights recent advances in programmable synthetic biology tools based on biosensors, CRISPR-Cas, and RNA devices for developing microbial cell factories that have the potential to be utilized for energy biotechnology.

Transmission Dynamics of the Delta Variant of SARS-CoV-2 Infections in South Korea.

BACKGROUND: The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the predominant variant worldwide at the time of this study. However, its transmission dynamics were unclear. METHODS: We analyzed 405 local cases infected with the Delta variant of SARS-CoV-2 and temporal patterns of viral shedding identified between 22 June and 31 July 2021 in Daejeon, South Korea. RESULTS: Overall, 20% were presymptomatic at the time of epidemiological investigation. We identified 6 clustered outbreaks, and all were associated with indoor facilities. In 23 household contacts, the secondary attack rate was 63%. We estimated the mean serial interval as 3.26 days (95% credible interval, 2.92-3.60), and 15% (95% confidence interval, 13%-18%) of cases seeded 80% of all local transmission. Analysis of the nasopharyngeal swab samples identified virus shedding from the presymptomatic cases and the highest viral load was observed 2 days after symptom onset. CONCLUSIONS: Our findings suggest that the Delta variant is highly transmissible in indoor settings and households. Rapid contact tracing, isolation of the asymptomatic contacts, strict adherence to public health measures, and increased uptake of coronavirus disease 2019 (COVID-19) vaccination, including booster doses, are needed to reduce community transmission of the Delta variant.

Ca2+ and Myosin Cycle States Work as Allosteric Effectors of Troponin Activation.

In cardiac muscle, troponin (Tn) and tropomyosin inhibit actin and myosin interactions through the steric blocking of myosin binding to F-actin. Ca2+ binding to Tn C modulates this inhibition. Thin filaments become activated upon Ca2+ binding, which enables strong binding of myosin with a concomitant release of ATP hydrolysis products and level arm swinging responsible for force generation. Despite this level of description, the current cross-bridge cycle model does not fully define the structural events that take place within Tn during combinatorial myosin and Ca2+ interventions. Here, we studied conformational changes within Tn bound to F-actin and tropomyosin by fluorescence lifetime imaging combined with Förster resonance energy transfer. Fluorescent dye molecules covalently bound to the Tn C C-lobe and Tn I C-terminal domain report Ca2+- and myosin-induced activation of Tn. Reconstituted thin filaments were deposited on a myosin-coated surface similar to an in vitro motility assay setup without filament sliding involved. Under all the tested conditions, Ca2+ was responsible for the most significant changes in Tn activation. Rigor myosin activated Tn at subsaturated Ca2+ conditions but not to the degree seen in thin filaments with Ca2+. ATP-γ-S did not affect Tn activation significantly; however, blebbistatin induced significant activation at subsaturating Ca2+ levels. The relation between the extent of Tn activation and its conformational flexibility suggests that active/inactive Tn states coexist in different proportions that depend on the combination of effectors. These results satisfy an allosteric activation model of the thin filament as a function of Ca2+ and the myosin catalytic cycle state.

Single-molecule analysis and lifetime estimates of heterogeneous low-count-rate time-correlated fluorescence data.

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.

Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ.

We report the first optical recordings of action potentials, in single trials, from one or a few (approximately 1-2 microm) mammalian nerve terminals in an intact in vitro preparation, the mouse neurohypophysis. The measurements used two-photon excitation along the "blue" edge of the two-photon absorption spectrum of di-3-ANEPPDHQ (a fluorescent voltage-sensitive naphthyl styryl-pyridinium dye), and epifluorescence detection, a configuration that is critical for noninvasive recording of electrical activity from intact brains. Single-trial recordings of action potentials exhibited signal-to-noise ratios of approximately 5:1 and fractional fluorescence changes of up to approximately 10%. This method, by virtue of its optical sectioning capability, deep tissue penetration, and efficient epifluorescence detection, offers clear advantages over linear, as well as other nonlinear optical techniques used to monitor voltage changes in localized neuronal regions, and provides an alternative to invasive electrode arrays for studying neuronal systems in vivo.