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  1. Abstract

    CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategymore »to be applied to improve the efficacy of other genome-editing proteins.

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  2. Free, publicly-accessible full text available February 1, 2023
  3. Free, publicly-accessible full text available March 28, 2023
  4. Chiral amines can be made by insertion of a carbene into an N–H bond using two-catalyst systems that combine a transition metal-based carbene-transfer catalyst and a chiral proton-transfer catalyst to enforce stereocontrol. Haem proteins can effect carbene N–H insertion, but asymmetric protonation in an active site replete with proton sources is challenging. Here we describe engineered cytochrome P450 enzymes that catalyse carbene N–H insertion to prepare biologically relevant α-amino lactones with high activity and enantioselectivity (up to 32,100 total turnovers, >99% yield and 98% e.e.). These enzymes serve as dual-function catalysts, inducing carbene transfer and promoting the subsequent proton transfer with excellent stereoselectivity in a single active site. Computational studies uncover the detailed mechanism of this new-to-nature enzymatic reaction and explain how active-site residues accelerate this transformation and provide stereocontrol.
  5. The properties of topological systems are inherently tied to their dimensionality. Indeed, higher-dimensional periodic systems exhibit topological phases not shared by their lower-dimensional counterparts. On the other hand, aperiodic arrays in lower-dimensional systems (e.g., the Harper model) have been successfully employed to emulate higher-dimensional physics. This raises a general question on the possibility of extended topological classification in lower dimensions, and whether the topological invariants of higher-dimensional periodic systems may assume a different meaning in their lower-dimensional aperiodic counterparts. Here, we demonstrate that, indeed, for a topological system in higher dimensions one can construct a one-dimensional (1D) deterministic aperiodic counterpart which retains its spectrum and topological characteristics. We consider a four-dimensional (4D) quantized hexadecapole higher-order topological insulator (HOTI) which supports topological corner modes. We apply the Lanczos transformation and map it onto an equivalent deterministic aperiodic 1D array (DAA) emulating 4D HOTI in 1D. We observe topological zero-energy zero-dimensional (0D) states of the DAA—the direct counterparts of corner states in 4D HOTI and the hallmark of the multipole topological phase, which is meaningless in lower dimensions. To explain this paradox, we show that higher-dimension invariant, the multipole polarization, retains its quantization in the DAA, yet changes its meaning by becomingmore »a nonlocal correlator in the 1D system. By introducing nonlocal topological phases of DAAs, our discovery opens a direction in topological physics. It also unveils opportunities to engineer topological states in aperiodic systems and paves the path to application of resonances associates with such states protected by nonlocal symmetries.« less
  6. Abstract

    Improved understanding of the effects of meteorological conditions on the transmission of SARS-CoV-2, the causative agent for COVID-19 disease, is needed. Here, we estimate the relationship between air temperature, specific humidity, and ultraviolet radiation and SARS-CoV-2 transmission in 2669 U.S. counties with abundant reported cases from March 15 to December 31, 2020. Specifically, we quantify the associations of daily mean temperature, specific humidity, and ultraviolet radiation with daily estimates of the SARS-CoV-2 reproduction number (Rt) and calculate the fraction ofRtattributable to these meteorological conditions. Lower air temperature (within the 20–40 °C range), lower specific humidity, and lower ultraviolet radiation were significantly associated with increasedRt. The fraction ofRtattributable to temperature, specific humidity, and ultraviolet radiation were 3.73% (95% empirical confidence interval [eCI]: 3.66–3.76%), 9.35% (95% eCI: 9.27–9.39%), and 4.44% (95% eCI: 4.38–4.47%), respectively. In total, 17.5% ofRtwas attributable to meteorological factors. The fractions attributable to meteorological factors generally were higher in northern counties than in southern counties. Our findings indicate that cold and dry weather and low levels of ultraviolet radiation are moderately associated with increased SARS-CoV-2 transmissibility, with humidity playing the largest role.