CRISPR/Cpf1-mediated DNA-free plant genome editing

Cpf1, a type V CRISPR effector, recognizes a thymidine-rich protospacer-adjacent motif and induces cohesive double-stranded breaks at the target site guided by a single CRISPR RNA (crRNA). Here we show that Cpf1 can be used as a tool for DNA-free editing of plant genomes. We describe the delivery of recombinant Cpf1 proteins with in vitro transcribed or chemically synthesized target-specific crRNAs into protoplasts isolated from soybean and wild tobacco. Designed crRNAs are unique and do not have similar sequences (≤3 mismatches) in the entire soybean reference genome. Targeted deep sequencing analyses show that mutations are successfully induced in FAD2 paralogues in soybean and AOC in wild tobacco. Unlike SpCas9, Cpf1 mainly induces various nucleotide deletions at target sites. No significant mutations are detected at potential off-target sites in the soybean genome. These results demonstrate that Cpf1-crRNA complex is an effective DNA-free genome-editing tool for plant genome editing. © 2017 The Author(s)384

Piperlongumine as a potential activator of AMP-activated protein kinase in HepG2 cells

<div><p>AMP-activated protein kinase (AMPK) is a key regulator of fatty acid biosynthesis and fatty acid oxidation throughout the body. Piperlongumine (PL) isolated from <i>Piper longum</i> (L.) was shown to potently upregulate activation of AMPK via phosphorylation and inactivation of acetyl-CoA carboxylases in cultured HepG2 cells, presumably enhancing the transfer of fatty acids into mitochondrial cells by inhibiting malonyl-CoA production. PL showed cytotoxicity on HepG2 cell growth at the concentration of 5 μM of PL, while more than 80% of HepG2 cells were survived at the concentration of 2 μM of PL. Overall, the results of this study indicate that PL activates AMPK phosphorylation and possesses cytotoxicity in HepG2 cells.</p></div

Author Correction: Precision genome engineering through adenine base editing in plants

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A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19−20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTM system and unique EcoRI/XhoI sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant. © 2016 Institute of Botany, Chinese Academy of Science19121sciescopu

Precision genome engineering through adenine base editing in plants

The recent development of adenine base editors (ABEs) has enabled efficient and precise A-to-G base conversions in higher eukaryotic cells. Here, we show that plant-compatible ABE systems can be successfully applied to protoplasts of Arabidopsis thaliana and Brassica napus through transient transfection, and to individual plants through Agrobacterium-mediated transformation to obtain organisms with desired phenotypes. Targeted, precise A-to-G substitutions generated a single amino acid change in the FT protein or mis-splicing of the PDS3 RNA transcript, and we could thereby obtain transgenic plants with late-flowering and albino phenotypes, respectively. Our results provide ‘proof of concept’ for in planta ABE applications that can lead to induced neo-functionalization or altered mRNA splicing, opening up new avenues for plant genome engineering and biotechnology. © 2018 The Author(s1

Generation of early-flowering Chinese cabbage (Brassica rapa spp. pekinensis) through CRISPR/Cas9-mediated genome editing

The CRISPR system enables us to induce precisely targeted mutations in a plant genome. The widely used CRISPR system is composed of a Cas9 protein derived from Streptococcus pyogenes (SpCas9) and a target site-specific guide RNA. In this study, we successfully generated the early-flowering Chinese cabbage (Brassica rapa spp. pekinensis), which is one of the most important vegetables in the world. To generate early-flowering B. rapa without requiring vernalization, we designed seven guide RNAs which target B. rapa homologous genes to the Arabidopsis thaliana FLOWERING LOCUS C (FLC). We first examined the indel mutation efficacy of the designed guide RNAs in protoplasts isolated from young leaves of Kenshin (an inbred line of B. rapa). After selecting four guide RNAs, genome-edited plants were established by delivering the plant binary vectors harboring SpCas9 along with respective guide RNAs into B. rapa hypocotyl explants. In the T-0 generation, we found BraFLC2 and BraFLC3 double knockout lines with the indel efficiency of 97.7% and 100%, respectively. The simultaneous mutations of both BraFLC2 and BraFLC3 were inherited in T-1 generations with 100% of indel efficiency. The edited lines obtained showed an early-flowering phenotype that did not depend on vernalization. This study provides a practical gene-editing protocol for Chinese cabbage and verifies the function of its multi-copy BraFLC genes.

Wafer-Scale Production of Two-Dimensional Tin Monoselenide: Expandable Synthetic Platform for van der Waals Semiconductor-Based Broadband Photodetectors

Copyright © 2023 American Chemical Society. A synthetic platform for industrially applicable two-dimensional (2D) semiconductors that addresses the paramount issues associated with large-scale production, wide-range photosensitive materials, and oxidative stability has not yet been developed. In this study, we attained the 6 in. scale production of 2D SnSe semiconductors with spatial homogeneity using a rational synthetic platform based on the thermal decomposition of solution-processed single-source precursors. The long-range structural and chemical homogeneities of the 2D SnSe layers are manifested using comprehensive spectroscopic analyses. Furthermore, the capability of the SnSe-based photodetectors for broadband photodetection is distinctly verified. The photoresponsivity and detectivity of the SnSe-based photodetectors are 5.89 A W-1 and 1.8 × 1011 Jones at 532 nm, 1.2 A W-1 and 3.7 × 1010 Jones at 1064 nm, and 0.14 A W-1 and 4.3 × 109 Jones at 1550 nm, respectively. The minimum rise times for the 532 and 1064 nm lasers are 62 and 374 μs, respectively. The photoelectrical analysis of the 5 × 5 SnSe-based photodetector array reveals 100% active devices with 95.06% photocurrent uniformity. We unequivocally validated that the air and thermal stabilities of the photocurrent yielded from the SnSe-based photodetector are determined to be >30 d in air and 160 °C, respectively, which are suitable for optoelectronic applications. © 2023 American Chemical Society.11Nsciescopu