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

In Supplementary Fig. 1b originally published with this Brief Communication, the DNA sequence of nickase Cas9 was incorrect; this has now been amended.

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.

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.

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 Gateway(TM) 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.

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

AMP-activated protein kinase (AMPK) is a key regulator of fatty acid biosynthesis and fatty acid oxidation throughout the body. Piperlongumine (PL) isolated from Piper longum (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.