Effect of Takadiastase on the Extraction of Biotin from Plant-Based Foods.

This study aimed to evaluate the extraction of biotin from plant-based foods using takadiastase in combination with autoclave- or ultrasound-assisted extraction. In cereals, vegetables, and mushrooms, autoclave-assisted enzymatic extraction obtained higher analytical values compared with autoclave-assisted extraction. However, in legumes and nuts, ultrasound-assisted enzymatic extraction obtained higher biotin content compared with either autoclave- or ultrasound-assisted extraction.

Identification of optimal adenine and cytosine base editors for genome editing in Arabidopsis and soybean.

Base editors, including adenine base editors (ABEs) and cytosine base editors (CBEs), are widely used in numerous organisms to introduce site-specific sequence modifications in genomic DNA without causing double-strand breaks (DSBs). However, these editors exhibit low editing efficiencies, particularly in dicot plants, thereby limiting their application in dicot plant genome engineering. In this study, we assessed the editing efficiencies of various base editors to identify those optimal for base editing in dicot plants.

Autophagy induction enhances homologous recombination-associated CRISPR-Cas9 gene editing.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9)-based gene editing via homologous recombination (HR) enables precise gene correction and insertion. However, its low efficiency poses a challenge due to the predominance of nonhomologous end-joining during DNA repair processes. Although numerous efforts have been made to boost HR efficiency, there remains a critical need to devise a novel method that can be universally applied across cell types and in vivo animals, which could ultimately facilitate therapeutic treatments.

Impaired chaperone-mediated autophagy leads to abnormal SORT1 (sortilin 1) turnover and CES1-dependent triglyceride hydrolysis.

SORT1 (sortilin 1), a member of the the Vps10 (vacuolar protein sorting 10) family, is involved in hepatic lipid metabolism by regulating very low-density lipoprotein (VLDL) secretion and facilitating the lysosomal degradation of CES1 (carboxylesterase 1), crucial for triglyceride (TG) breakdown in the liver. This study explores whether SORT1 is targeted for degradation by chaperone-mediated autophagy (CMA), a selective protein degradation pathway that directs proteins containing KFERQ-like motifs to lysosomes via LAMP2A (lysosomal-associated membrane protein 2A). Silencing LAMP2A or HSPA8/Hsc70 with siRNA increased cytosolic SORT1 protein levels.

Production of the antimalarial drug precursor amorphadiene by microbial terpene synthase-like from the moss Sanionia uncinata.

The microbial terpene synthase-like of the moss Sanionia uncinata displays the convergent evolution of a rare plant metabolite amorpha-4,11-diene synthesis. Despite increasing demand for the exploration of biological resources, the diversity of natural compounds synthesized by organisms inhabiting various climates remains largely unexplored. This study focuses on the moss Sanionia uncinata, known as a predominant species within the polar climates of the Antarctic Peninsula, to systematically explore its metabolic profile both in-field and in controlled environments.

Phenotypic characterization of pre-harvest sprouting resistance mutants generated by the CRISPR/Cas9-geminiviral replicon system in rice.

Pre-harvest sprouting is a critical phenomenon involving germination of seeds in the mother plant before harvest under relative humid conditions and reduced dormancy. In this paper, we generated HDR mutant lines with one region SNP (C/T) and an insertion of 6 bp (GGT/GGTGGCGGC) in OsERF1 genes for pre-harvest sprouting (PHS) resistance using CRISPR/Cas9 and a geminiviral replicon system. The incidence of HDR was 2.

Life under the snow: A year-round transcriptome analysis of Antarctic mosses in natural habitats provides insight into the molecular adaptation of plants under extreme environment.

Mosses are vital components of ecosystems, exhibiting remarkable adaptability across diverse habitats from deserts to polar ice caps. Sanionia uncinata (Hedw.) Loeske, a dominant Antarctic moss survives extreme environmental condition through perennial lifecycles involving growth and dormancy alternation.

Chaperone-mediated autophagy dysregulation during aging impairs hepatic fatty acid oxidation via accumulation of NCoR1.

Objective: Alterations in lipid metabolism are associated with aging and age-related diseases. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process involved in specific protein degradation. Heat shock cognate 71 kDa protein (Hsc70) recognizes cytosolic proteins with KFERQ motif and allows them to enter the lysosome via lysosome-associated membrane glycoprotein 2 isoform A (LAMP2A).

Biochemical Characterization of Orange-Colored Rice Calli Induced by Target Mutagenesis of Gene.

We generated an orange-colored (OC) rice callus line by targeted mutagenesis of the orange gene () using the CRISPR-Cas9 system. The OC line accumulated more lutein, -carotene, and two -carotene isomers compared to the WT callus line. We also analyzed the expression levels of carotenoid biosynthesis genes by qRT-PCR.

Transcriptome and Metabolite Profiling of Tomato SGR-Knockout Null Lines Using the CRISPR/Cas9 System.

Stay-green 1 (SGR1) protein is a critical regulator of chlorophyll degradation and senescence in plant leaves; however, the functions of tomato SGR1 remain ambiguous. Here, we generated an SGR1-knockout (KO) null line via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-mediated gene editing and conducted RNA sequencing and gas chromatography−tandem mass spectrometry analysis to identify the differentially expressed genes (DEGs). Solanum lycopersicum SGR1 (SlSGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid levels than those in the wild-type (WT) fruit.

Identification and Characterization of () and Genes by CRISPR/Cas9-Targeted Mutagenesis in Chinese Cabbage ( L.).

The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, () , , and (both and double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9-16 genetic edited lines of each mutant were obtained.

The two clock proteins CCA1 and LHY activate VIN3 transcription during vernalization through the vernalization-responsive cis-element.

Vernalization, a long-term cold-mediated acquisition of flowering competence, is critically regulated by VERNALIZATION INSENSITIVE 3 (VIN3), a gene induced by vernalization in Arabidopsis. Although the function of VIN3 has been extensively studied, how VIN3 expression itself is upregulated by long-term cold is not well understood. In this study, we identified a vernalization-responsive cis-element in the VIN3 promoter, VREVIN3, composed of a G-box and an evening element (EE).

Arabidopsis ATXR2 represses de novo shoot organogenesis in the transition from callus to shoot formation.

Plants exhibit high regenerative capacity, which is controlled by various genetic factors. Here, we report that ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2) controls de novo shoot organogenesis by regulating auxin-cytokinin interaction. The auxin-inducible ATXR2 Trithorax Group (TrxG) protein temporally interacts with the cytokinin-responsive type-B ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) at early stages of shoot regeneration.

AC-motif: a DNA motif containing adenine and cytosine repeat plays a role in gene regulation.

I-motif or C4 is a four-stranded DNA structure with a protonated cytosine:cytosine base pair (C+:C) found in cytosine-rich sequences. We have found that oligodeoxynucleotides containing adenine and cytosine repeats form a stable secondary structure at a physiological pH with magnesium ion, which is similar to i-motif structure, and have named this structure 'adenine:cytosine-motif (AC-motif)'. AC-motif contains C+:C base pairs intercalated with putative A+:C base pairs between protonated adenine and cytosine.

High-purity production and precise editing of DNA base editing ribonucleoproteins.

Ribonucleoprotein (RNP) complex-mediated base editing is expected to be greatly beneficial because of its reduced off-target effects compared to plasmid- or viral vector-mediated gene editing, especially in therapeutic applications. However, production of recombinant cytosine base editors (CBEs) or adenine base editors (ABEs) with ample yield and high purity in bacterial systems is challenging. Here, we obtained highly purified CBE/ABE proteins from a human cell expression system and showed that CBE/ABE RNPs exhibited different editing patterns (i.

Analysis of NHEJ-Based DNA Repair after CRISPR-Mediated DNA Cleavage.

Genome editing using CRISPR-Cas9 nucleases is based on the repair of the DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed repair (HDR), non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) pathways. Among these, it is thought that the NHEJ pathway is dominant and occurs throughout a cell cycle.

In vivo gene editing via homology-independent targeted integration for adrenoleukodystrophy treatment.

Adrenoleukodystrophy (ALD) is caused by various pathogenic mutations in the X-linked ABCD1 gene, which lead to metabolically abnormal accumulations of very long-chain fatty acids in many organs. However, curative treatment of ALD has not yet been achieved. To treat ALD, we applied two different gene-editing strategies, base editing and homology-independent targeted integration (HITI), in ALD patient-derived fibroblasts.

Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens.

Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number.

Mitochondrial genome of the Antarctic microalga KSF0127 (Chlorellaceae, Trebouxiophyceae).

We report the first mitochondrial genome of the Antarctic microalga KSF0127. The circular mitochondrial genome was 67,923 bp in length and contained 45 protein-coding genes, one ribosomal RNA gene, and 60 transfer RNA genes. The phylogenetic tree was constructed with eight previously reported mitogenome sequences and showed the phylogenetic position of KSF0127 within the Chlorellaceae family.

Transcriptomic and physiological analysis of OsCAO1 knockout lines using the CRISPR/Cas9 system in rice.

The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported.

Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.

We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries.

Generation and Transcriptome Profiling of Slr1-d7 and Slr1-d8 Mutant Lines with a New Semi-Dominant Dwarf Allele of Using the CRISPR/Cas9 System in Rice.

The rice gene encodes the DELLA protein, and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated.