Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants.

BACKGROUND: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells. RESULTS: To systematically investigate TALE-DdCBE architectures and editing rules, we established a ß-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. CONCLUSIONS: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.

Development of TALE-adenine base editors in plants.

Base editors enable precise nucleotide changes at targeted genomic loci without requiring double-stranded DNA breaks or repair templates. TALE-adenine base editors (TALE-ABEs) are genome editing tools, composed of a DNA-binding domain from transcription activator-like effectors (TALEs), an engineered adenosine deaminase (TadA8e), and a cytosine deaminase domain (DddA), that allow A•T-to-G•C editing in human mitochondrial DNA. However, the editing ability of TALE-ABEs in plants apart from chloroplast DNA has not been described, so far, and the functional role how DddA enhances TadA8e is still unclear. We tested a series of TALE-ABEs with different deaminase fusion architectures in Nicotiana benthamiana and rice. The results indicate that the double-stranded DNA-specific cytosine deaminase DddA can boost the activities of single-stranded DNA-specific deaminases (TadA8e or APOBEC3A) on double-stranded DNA. We analysed A•T-to-G•C editing efficiencies in a ß-glucuronidase reporter system and showed precise adenine editing in genomic regions with high product purity in rice protoplasts. Furthermore, we have successfully regenerated rice plants with A•T-to-G•C mutations in the chloroplast genome using TALE-ABE. Consequently, TALE-adenine base editors provide alternatives for crop improvement and gene therapy by editing nuclear or organellar genomes.

Sea urchin-like amorphous MgNiCo mixed metal hydroxide nanoarrays for efficient overall water splitting under industrial electrolytic conditions.

Exploring amorphous mixed transition metal hydroxide electrocatalysts with high performance and stability for overall water splitting is a difficult challenge under industrial electrolytic conditions. Herein, a sea urchin-like amorphous MgNiCo hydroxide (MgxNi1-xCo-OH, 0 < x < 1), self-assembled from nanowire arrays, is synthesized by the hydrothermal process. The synergistic effect between Mg and Ni/Co adjusts their crystal structure and morphology, which can improve the inherent activity and provide more active sites. Benefiting from the favorable structural features, Mg0.5Ni0.5Co-OH exhibits superior electrocatalytic oxygen and hydrogen evolution reaction (OER and HER) activity with a low overpotential of 277 and 110 mV (10 mA cm-2) in 1 M KOH at 25 °C. Furthermore, overpotentials of 239 and 197 mV are required to achieve a current density of 50 mA cm-2 for the OER and HER under simulated industrial electrolysis conditions (5 M KOH at 65 °C). Notably, Mg0.5Ni0.5Co-OH remarkably accelerates water splitting with a low voltage of 1.938 and 1.699 V for 50 mA cm-2 in 1 M KOH at 25 °C and 5 M KOH at 65 °C, respectively. This work presents a novel amorphous strategy to design and construct sea urchin-like mixed metal hydroxide bifunctional efficient electrocatalysts for industrial applications.

Regulating the thermal conductivity of monolayer MnPS3 by a magnetic phase transition.

In this study, based on ab initio calculations and the phonon Boltzmann transport equation, we found that magnetic phase transitions can lead to a significant change in the thermal conductivity of monolayer MnPS3. Around the Néel temperature (78 K) with the antiferromagnetic-paramagnetic (AFM-PM) phase transition, its thermal conductivity increases from 14.89 W mK-1 (AFM phase) to 103.21 W mK-1 (PM phase). Below 78 K, the thermal conductivity of monolayer MnPS3 can be doubled by applying a magnetic field of 4 T, this value has been reported in a previous experiment for the antiferromagnetic-ferromagnetic (AFM-FM) phase transition. Above 78 K, the thermal conductivity of PM phase can be greatly reduced through the PM-AFM magnetic phase transition. In addition to the value of thermal conductivity, the relative contribution ratio between acoustic and optical modes changes with different magnetic phases. The subsequent analyses demonstrate that this regulation originates from the change in lattice parameter, bonding interaction and phonon anharmonicity. In addition, the different effect on the thermal conductivity between the FM and AFM phases was identified by comparing the corresponding phonon scattering characteristics. This study should shed light on the understanding of phonon thermal conductivity in 2D magnets, and provide a practical method for the realization of 2D thermal switching devices, which would enable a broad range of novel applications including energy conversion and thermal management.

Removing the major allergen Bra j I from brown mustard (Brassica juncea) by CRISPR/Cas9.

Food allergies are a major health issue worldwide. Modern breeding techniques such as genome editing via CRISPR/Cas9 have the potential to mitigate this by targeting allergens in plants. This study addressed the major allergen Bra j I, a seed storage protein of the 2S albumin class, in the allotetraploid brown mustard (Brassica juncea). Cotyledon explants of an Indian gene bank accession (CR2664) and the German variety Terratop were transformed using Agrobacterium tumefaciens harboring binary vectors with multiple single guide RNAs to induce either large deletions or frameshift mutations in both Bra j I homoeologs. A total of 49 T0 lines were obtained with up to 3.8% transformation efficiency. Four lines had large deletions of 566 up to 790 bp in the Bra j IB allele. Among 18 Terratop T0 lines, nine carried indels in the targeted regions. From 16 analyzed CR2664 T0 lines, 14 held indels and three had all four Bra j I alleles mutated. The majority of the CRISPR/Cas9-induced mutations were heritable to T1 progenies. In some edited lines, seed formation and viability were reduced and seeds showed a precocious development of the embryo leading to a rupture of the testa already in the siliques. Immunoblotting using newly developed Bra j I-specific antibodies revealed the amount of Bra j I protein to be reduced or absent in seed extracts of selected lines. Removing an allergenic determinant from mustard is an important first step towards the development of safer food crops.

Novel two-dimensional beta-XTe (X = Ge, Sn, Pb) as promising room-temperature thermoelectrics.

In this paper, we designed novel low-symmetry two-dimensional (2D) structures based on conventional XTe (X = Ge, Sn, Pb) thermoelectrics with large average atomic mass. The first-principles calculations combined with Boltzmann transport theory show that the beta-XTe exhibit good stability, high electron carrier mobility, and ultralow ΚL. The subsequent analyses show that the ultralow ΚL stems from the coexistence of resonant bonding, weak bonding, and lone-pair electrons in beta-XTe, which leads to large anharmonicities. On the other hand, the lowest energy conduction band of beta-GeTe and beta-SnTe show the convergence of the low-lying Æ© band, which is the source of the high-power factor in the two systems. The calculated maximum ZT of beta-XTe (X = Ge, Sn, Pb) are 3.08, 1.60, and 0.57 at 300 K, respectively, which is significantly greater than that of the previously reported high-symmetry 2D alpha-XTe and the commercial thermoelectrics. We hope that this work can provide important guidance for the development of thermoelectric materials.

The thermoelectric properties of α-XP (X = Sb and Bi) monolayers from first-principles calculations.

Thermoelectric (TE) materials as one of the effective solutions to the energy crisis are gaining more and more interest owing to their capability to generate electricity from waste heat without generating air pollution. In this work, the TE properties of α-XP monolayers such as the stability, electronic structure, electrical and phonon transport were thoroughly studied in combination with the first-principles calculations and Boltzmann transport equations. We found that α-SbP and α-BiP have indirect bandgaps of 0.85 eV and 0.73 eV, respectively, which are suitable for thermoelectric materials. Furthermore, due to the multiple valleys at the energy band edges and the high carrier mobility, α-XP possesses both large Seebeck coefficients and high electrical conductivities. It is also found that the lattice thermal conductivity of α-BiP is smaller than that of α-SbP due to lower phonon frequencies, smaller phonon group velocities, larger Grüneisen parameters and higher phonon relaxation times. High TE performance was achieved with the ZT values reaching 4.59 (for α-BiP at 500 K) and 1.34 (for α-SbP at 700 K). Our results quantify α-XP monolayers as promising candidates for building outstanding thermoelectric devices.

Promising thermoelectric candidate based on a CaAs3 monolayer: A first principles study.

The CaAs3 monolayer is a newly predicted two-dimensional material with attractive properties, such as a moderate direct bandgap, high carrier mobility, prominent visible-light absorption, etc. To evaluate its potential applications in thermoelectric (TE) fields, herein, the thermoelectric properties of CaAs3 monolayers were comprehensively investigated by a first-principles method in combination with Boltzmann transport theory. Our calculated results indicate that the CaAs3 monolayer has an exceptionally low lattice thermal conductivity of 0.44 W m-1 K-1 at 300 K, mainly because of the small group velocity and strong phonon-phonon scattering. The CaAs3 monolayer also exhibits a high power factor due to the large Seebeck coefficient and electrical conductivity. Therefore, large ZT values of 1.72/1.58 were achieved for the n-type/p-type CaAs3 monolayer at 800 K. Compared with conventional 2D TE materials, the CaAs3 monolayer does not contain expensive heavy elements, which is beneficial for its practical applications as a TE material. Our results qualify the CaAs3 monolayer as a promising candidate for building excellent 2D TE devices.

Effects of out-of-plane strains and electric fields on the electronic structures of graphene/MTe (M = Al, B) heterostructures.

Contacts between graphene and two-dimensional (2D) semiconductors have been widely investigated because of their tunable Schottky barrier height (SBH) by means of applied out-of-plane strains, electric fields, etc. Here, based on first-principles calculations, we study the effects of out-of-plane strains (a tensile or compressive strain) and electric fields on the electronic structures of graphene/MTe (M = Al, B) heterostructures. The calculated results indicate that p-type Schottky barriers are formed at the graphene/AlTe and graphene/BTe interfaces with 0.72 and 0.49 eV, respectively. The increase in the interlayer distances (tensile strains) between graphene and MTe can induce a transition from a p-type to n-type Schottky contact. On the other hand, the decrease in the interlayer distances (compressive strains) can transform graphene/MTe into semiconductors, which originates from graphene/MTe with a large compressive strain that makes the two carbon sublattices inequivalent, inducing a band gap. In addition, the applied electric fields can modulate effectively the contact formation (a Schottky or Ohmic contact) and the doping of graphene in graphene/MTe heterostructures. Our study suggests two facile methods to tune the electronic properties of graphene/MTe heterostructures and offer a possibility for graphene/MTe heterostructure-based electronic devices.

Boosting activity of high-fidelity CRISPR/Cas9 variants using a tRNAGln-processing system in human cells.

CRISPR/Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. High-fidelity Cas9 variants have been identified; however, they often have reduced activity, constraining their utility, which presents a major challenge for their use in research applications and therapeutics. Here we developed a tRNAGln-processing system to restore the activity of multiple high-fidelity Cas9 variants in human cells, including SpCas9-HF1, eSpCas9, and xCas9. Specifically, acting on previous observations that small guide RNAs (sgRNAs) harboring an extra A or G (A/G) in the first 5' nucleotide greatly affect the activity of high-fidelity Cas9 variants and that tRNA-sgRNA fusions improve Cas9 activity, we investigated whether a GN20 sgRNA fused to different tRNAs (G-tRNA-N20) could restore the activity of SpCas9 variants in human cells. Using flow cytometry, a T7E1 assay, deep sequencing-based DNA cleavage activity assays, and HEK-293 cells, we observed that a tRNAGln-sgRNA fusion system enhanced the activity of Cas9 variants, which could be harnessed for efficient correction of a pathogenic mutation in the retinoschisin 1 (RS1) gene, resulting in 6- to 8-fold improved Cas9 activity. We propose that the tRNA-processing system developed here specifically for human cells could facilitate high-fidelity Cas9-mediated human genome-editing applications.

Tunable Electric Properties of Bilayer α-GeTe with Different Interlayer Distances and External Electric Fields.

Based on first-principle calculations, the stability, electronic structure, optical absorption, and modulated electronic properties by different interlayer distances or by external electric fields of bilayer α-GeTe are systemically investigated. Results show that van der Waals (vdW) bilayer α-GeTe has an indirect band structure with the gap value of 0.610 eV, and α-GeTe has attractively efficient light harvesting. Interestingly, along with the decrease of interlayer distances, the band gap of bilayer α-GeTe decreases linearly, due to the enhancement of interlayer vdW interaction. In addition, band gap transition is originated from the electric field-induced near free-electron gas (NFEG) under the application of positive electrical fields. However, when the negative electric fields are applied, there is no NFEG. On account of these characteristics of bilayer α-GeTe, a possible data storage device has been designed. These results indicate that bilayer α-GeTe has a potential to work in new electronic and optoelectronic devices.

Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A.

Base editors (BEs) have been used to create C-to-T substitutions in various organisms. However, editing with rat APOBEC1-based BE3 is limited to a 5-nt sequence editing window and is inefficient in GC contexts. Here, we show that a base editor fusion protein composed of Cas9 nickase and human APOBEC3A (A3A-PBE) converts cytidine to thymidine efficiently in wheat, rice and potato with a 17-nucleotide editing window at all examined sites, independent of sequence context.

Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion.

Nucleotide base editors in plants have been limited to conversion of cytosine to thymine. Here, we describe a new plant adenine base editor based on an evolved tRNA adenosine deaminase fused to the nickase CRISPR/Cas9, enabling A•T to G•C conversion at frequencies up to 7.5% in protoplasts and 59.1% in regenerated rice and wheat plants. An endogenous gene is also successfully modified through introducing a gain-of-function point mutation to directly produce an herbicide-tolerant rice plant. With this new adenine base editing system, it is now possible to precisely edit all base pairs, thus expanding the toolset for precise editing in plants.

Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits.

GW2 is emerging as a key genetic determinant of grain weight in cereal crops; it has three homoeologs (TaGW2-A1, -B1 and -D1) in hexaploid common wheat (Triticum aestivum L.). Here, by analyzing the gene editing mutants that lack one (B1 or D1), two (B1 and D1) or all three (A1, B1 and D1) homoeologs of TaGW2, several insights are gained into the functions of TaGW2-B1 and -D1 in common wheat grain traits. First, both TaGW2-B1 and -D1 affect thousand-grain weight (TGW) by influencing grain width and length, but the effect conferred by TaGW2-B1 is stronger than that of TaGW2-D1. Second, there exists functional interaction between TaGW2 homoeologs because the TGW increase shown by a double mutant (lacking B1 and D1) was substantially larger than that of their single mutants. Third, both TaGW2-B1 and -D1 modulate cell number and length in the outer pericarp of developing grains, with TaGW2-B1 being more potent. Finally, TaGW2 homoeologs also affect grain protein content as this parameter was generally increased in the mutants, especially in the lines lacking two or three homoeologs. Consistent with this finding, two wheat end-use quality-related parameters, flour protein content and gluten strength, were considerably elevated in the mutants. Collectively, our data shed light on functional difference between and additive interaction of TaGW2 homoeologs in the genetic control of grain weight and protein content traits in common wheat, which may accelerate further research on this important gene and its application in wheat improvement.

Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases.

High-fidelity SpCas9 variants (eSpCas9 and SpCas9-HF1) have been engineered to reduce off-target effects. We found that changes in guide RNA length induced significant reductions in the editing activities of SpCas9 variants in plant cells. Single guide RNAs harboring precise, perfectly matched 20-nucleotide guide sequences are necessary for high on-target editing activities of eSpCas9 and SpCas9-HF1. Precise 20-nucleotide guide sequences derived from tRNA-sgRNA precursors enable robust on-target editing by these variants with enhanced specificity. Our work reveals an effective way of enhancing the use of the high-fidelity SpCas9 nucleases for efficient and precise genome engineering.