RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern.

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

Phase separation modulates the assembly and dynamics of a polarity-related scaffold-signaling hub.

Asymmetric cell division (ACD) produces morphologically and behaviorally distinct cells and is the primary way to generate cell diversity. In the model bacterium Caulobacter crescentus, the polarization of distinct scaffold-signaling hubs at the swarmer and stalked cell poles constitutes the basis of ACD. However, mechanisms involved in the formation of these hubs remain elusive. Here, we show that a swarmer-cell-pole scaffold, PodJ, forms biomolecular condensates both in vitro and in living cells via phase separation. The coiled-coil 4-6 and the intrinsically disordered regions are the primary domains that contribute to biomolecular condensate generation and signaling protein recruitment in PodJ. Moreover, a negative regulation of PodJ phase separation by the stalked-cell-pole scaffold protein SpmX is revealed. SpmX impedes PodJ cell-pole accumulation and affects its recruitment ability. Together, by modulating the assembly and dynamics of scaffold-signaling hubs, phase separation may serve as a general biophysical mechanism that underlies the regulation of ACD in bacteria and other organisms.

Creating RNA Specific C-to-U Editase from APOBEC3A by Separation of Its Activities on DNA and RNA Substrates.

APOBEC3A (A3A) is a cytidine deaminase involved in innate immune response and is able to catalyze deamination on both DNA and RNA substrates. It was used in creating the CRISPR-mediated base editor, but has since been held back due to its dual activities. On the other hand, it has been a challenge to separate A3A's dual activities in order to enable it for single-base RNA editors. Here we developed the reporter system for C-to-U RNA editing and employed rational design for mutagenesis to differentiate deaminase activities on RNA and DNA substrates to obtain an RNA-specific editase. Generation and examination of 23 previous A3A mutants showed their deamination activity on RNA was mostly abolished when their activity on DNA was impaired, with the exception of mutant N57Q that displayed an inverse change. We designed new mutations on Loops 1 and 7 based on A3A's crystal structure and found mutants H29R and Y132G had differential effects on catalytic activity on RNA and DNA substrates. In order to engineer an A3A with RNA-specific deaminase activity, we combined Y132G with mutations in Loop 1 or helix 6 by rational design. Two multipoint mutants, Y132G/K30R and Y132G/G188A/R189A/L190A, were successful in retaining high deaminase activity on RNA substrate while eliminating deaminase activity on DNA. We, for the first time, created novel human A3A variants with RNA-specific cytidine deaminase activity, providing insight into A3A's mechanism on substrate recognition and a new addition of a toolset to the creation of a RNA-specific C-to-U base editor.

Understanding the influence of Tween 80 on pullulan fermentation by Aureobasidium pullulans CGMCC1234.

In this paper, several new perspectives concerned with the effect of Tween 80 promoting pullulan production were presented. With the presence of Tween 80, the maximum pullulan yield increased by 41% (53.04 g/L). Meanwhile, the carbon source was consumed faster and the broth viscosity was higher. The lower final pH suggested that Tween 80 could protect the integrity of the mycelia. The dispersed filaments were not easily entangled with each other and less pellets were formed in the Tween 80 culture broth. FT-IR spectrum analysis indicated that the evaluated sample structure was coincided with commercial pullulan. The molecular weight of sample significantly dropped comparing with the control. The above findings indicated that Tween 80 facilitated the uptake of nutrient from surroundings to the microorganism and the release of pullulan into the extracellular fluid. These results were useful in better understanding the regulation and optimization of efficient pullulan fermentation.