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Genome‐editing using the CRISPR‐Cas9 system has the potential to substantially accelerate crop breeding. Since off‐target editing is one of problems, a reliable method for comprehensively detecting off‐target sites is needed. A number of in silico methods based on homology to on‐target sequence have been developed, however the prediction without false negative is still under discussion. In this study, we performed a SITE‐Seq analysis to predict potential off‐target sites. SITE‐Seq analysis is a comprehensive method that can detect double‐strand breaks in vitro. Furthermore, we developed a systematic method using SITE‐Seq in combination with web‐based Galaxy system (Galaxy for Cut Site Detection), which can perform reproducible analyses without command line operations. We conducted a SITE‐Seq analysis of a rice genome targeted by OsFH15 gRNA‐Cas9 as a model, and found 41 candidate off‐target sites in the annotated regions. Detailed amplicon‐sequencing revealed mutations at one off‐target site in actual genome‐edited rice. Since this off‐target site has an uncommon protospacer adjacent motif, it is difficult to predict using in silico methods alone. Therefore, we propose a novel off‐target assessment scheme for genome‐edited crops that combines the prediction of off‐target candidates by SITE‐Seq and in silico programs and the validation of off‐target sites by amplicon‐sequencing. [ABSTRACT FROM AUTHOR]

Single-nucleotide polymorphisms ( SNPs) are one of the main causes of evolution. The distribution of human SNPs, which were examined in detail genomewide, was analyzed. Three discrete databases of human SNPs were used for this analysis, and similar results were obtained from these databases. It was found that the distribution of the distance between SNPs was approximated by the power law, and the shape of the regions including SNPs had the so-called fractal structure. Although the reason why the distribution of SNPs obeys such a certain law of physics is unclear, a speculation was attempted in connection with the three-dimensional structure of human chromatin which has a fractal structure. [ABSTRACT FROM AUTHOR]

S treptomyces rochei 7434 AN4 carries three linear plasmids, p SLA2-L (211 kb), p SLA2- M (113 kb) and p SLA2- S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of p SLA2- L and p SLA2- M up to 3.1 kb from the ends and have homology to those of typical S treptomyces species. Mutant 2-39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of p SLA2-L from strain 51252, which carries only p SLA2- L, also resulted in terminal deletions in newly obtained mutants. The tap-tpg gene pair, which encodes a telomere-associated protein and a terminal protein for end patching, is located on p SLA2- L and p SLA2- M but has not hitherto been found on the chromosome. These results led us to the idea that the tap-tpg of p SLA2- L or p SLA2- M functions to maintain a linear chromosome in strain 7434 AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap-tpg of p SLA2- M. [ABSTRACT FROM AUTHOR]

Three types of mitosis, which are open, closed or semi-open mitosis, function in eukaryotic cells, respectively. The open mitosis involves breakage of the nuclear envelope before nuclear division, whereas the closed mitosis proceeds with an intact nuclear envelope. To understand the mechanism and significance of three types of mitotic division in eukaryotes, we investigated the process of semi-open mitosis, in which the nuclear envelope is only partially broken, in the fission yeast Schizosaccharomyces japonicus. In anaphase-promoting complex/cyclosome ( APC/C) mutants of Sz. japonicus, the nuclear envelope remained relatively intact during anaphase, resulting in impaired semi-open mitosis. As a suppressor of apc2 mutant, a mutation of Oar2, which was a 3-oxoacyl-[acyl carrier protein] reductase, was obtained. The level of the Oar2, which had two destruction-box motifs recognized by APC/C, was increased in APC/C mutants. Furthermore, the defective semi-open mitosis observed in an apc2 mutant was restored by mutated oar2 +. Based on these findings, we propose that APC/C regulates the dynamics of the nuclear envelope through degradation of Oar2 dependent on APC/C during the metaphase-to-anaphase transition of semi-open mitosis in Sz. japonicus. [ABSTRACT FROM AUTHOR]

Summary While bacteria such as Escherichia coli and Bacillus subtilis harbour a single circular chromosome, some freshwater cyanobacteria have multiple chromosomes per cell. The detailed mechanism(s) of cyanobacterial replication remains unclear. To elucidate the replication origin ( ori), form and synchrony of the multi-copy genome in freshwater cyanobacteria Synechococcus elongatus PCC 7942 we constructed strain S. 7942TK that can incorporate 5-bromo-2'-deoxyuridine (BrdU) into genomic DNA and analysed its de novo DNA synthesis. The uptake of BrdU was blocked under dark and resumed after transfer of the culture to light conditions. Mapping analysis of nascent DNA fragments using a next-generation sequencer indicated that replication starts bidirectionally from a single ori, which locates in the upstream region of the dnaN gene. Quantitative analysis of BrdU-labelled DNA and whole-genome sequence analysis indicated that the peak timing of replication precedes that of cell division and that replication is initiated asynchronously not only among cell populations but also among the multi-copy chromosomes. Our findings suggest that replication initiation is regulated less stringently in S. 7942 than in E. coli and B. subtilis. [ABSTRACT FROM AUTHOR]

Dry aging (DA) allows for the storage of meat without packaging at 0 to 3°C for several weeks. It enhances the production of pleasant flavors, tenderness, and juiciness in meat. Due to the long storage period and roles of indigenous microbiota in the maturation of several meat products, the microbiota of DA meat is of interest in terms of microbial contributions and food hygiene but has not yet been characterized in detail. This study identified the microbiota of pork loins during DA using culturing and culture-independent meta-16S rRNA gene sequencing and elucidated its characteristics. The amounts of free amino acids and profiles of aroma-active compounds were also monitored by high-performance liquid chromatography and gas chromatography, respectively. The meta-16S rRNA gene sequencing revealed that Pseudomonas spp. generally dominated the microbiota throughout DA; however, the culturing analysis showed marked changes in the species composition during DA. Acinetobacter spp. were the second most dominant bacteria before DA in the culture-independent analysis but became a minor population during DA. The cell numbers of yeasts showed an increased tendency during DA, and Debaryomyces hansenii was the only microorganism isolated from all meat samples throughout DA. Well-known foodborne pathogens were not observed in two microbiota analyses. The amounts of free amino acids were increased by DA, and the number of aroma-active compounds and their flavor dilution values markedly changed during DA. Most microbial isolates showed positive reactions with proteolytic and lipolytic activities, suggesting their contribution to tenderness and aroma production in DA meats., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)