Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using sixmodule concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/ in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms.

An unhealthy diet with excessive fat intake has often been claimed to induce not only obesity but also cognitive dysfunction in mammals; however, it is not known whether this is the case in zebrafish. Here, we investigated the effect of excessive fat in the diet on cognitive function and on gene expression in the telencephalon of zebrafish. Cognitive function, as measured by active avoidance test, was impaired by feeding of a high-fat diet compared with a control diet. In RNA sequencing analysis of the telencephalon, 97 genes were identified with a fold change in expression greater than 2 and a p-value less than 0.05 between the two diets. In quantitative real-time PCR analysis of the telencephalon, genes related to neuronal activity, anti-oxidative stress, blood-brain barrier function and amyloid-β degradation were found to be downregulated, whereas genes related to apoptosis and amyloid-β production were found to be upregulated, in the high-fat diet group, which are changes known to occur in mammals fed a high-fat diet. Collectively, these results are similar to those found in mammals, suggesting that zebrafish can serve as a suitable animal model in research into cognitive impairment induced by excessive fat in the diet.According to the World Health Organization, in December 2017 around 50 million people had dementia, and 10 million new cases emerge every year 1 . Dementia is a syndrome-usually of a chronic or progressive nature-in which there is deterioration in cognitive function 1 . Some research has shown a relationship between the development of cognitive impairment and life-style-related risk factors that are shared with other non-communicable diseases 1 . Obesity and unhealthy diet are two risk factors for dementia 1,2 . Excessive fat intake has often been claimed to induce obesity in humans and rodents 3,4 . Although numerous studies have been conducted to elucidate the effects of obesity and excessive dietary fat intake on cognitive function in rodents [5][6][7] , no such studies have been conducted in zebrafish (Danio rerio).The organs and tissues of zebrafish are similar in terms of structure and function to those of humans. In addition, these fish are amenable to genetic manipulation, breed readily in captivity, and can be inexpensively maintained, making them a useful model organism for investigating many human pathological conditions 8 . Several reports suggest that zebrafish are a suitable model organism for examining the mechanisms underlying lipid metabolism leading to obesity. For example, overexpression of the endogenous melanocortin antagonist agouti-related protein or the serine/threonine protein kinase Akt1 results in the development of adiposity in zebrafish 9,10 . Overfeeding and a high-fat (HF) diet induce body fat accumulation in zebrafish through pathophysiological pathways common with those underlying mammalian obesity; furthermore, green tea extract and fish oil, which have well-known anti-obesity activities, can cancel out these effects. Thus, zebrafish are a useful model of human...

Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos.

Post-intensive care syndrome (PICS) is characterized by several prolonged symptoms after critical care, including physical and cognitive dysfunctions as well as mental illness. In clinical practice, the long-term follow-up of PICS is initiated after patients have been discharged from the intensive care unit, and one of the approaches used is a PICS clinic. Although physical dysfunction and mental illness often present in combination, they have not yet been examined in detail in PICS patients. Grip strength is a useful physical examination for PICS, and is reported to be associated with mental status in the elderly. We herein investigated the relationship between grip strength and the mental status using data from our PICS clinic. We primarily aimed to analyze the correlation between grip strength and the Hospital Anxiety and Depression Scale (HADS) score. We also analyzed the association between grip strength and the EuroQol 5 Dimension (EQ5D) score as quality of life (QOL). Subjects comprised 133 patients who visited the PICS clinic at one month after hospital discharge between August 2019 and December 2020. Total HADS scores were 7 (4, 13) and 10 (6, 16) (p = 0.029) and EQ5D scores were 0.96 (0.84, 1) and 0.77 (0.62, 0.89) (p ≤ 0.0001) in the no walking disability group and walking disability group, respectively. Grip strength negatively correlated with HADS and EQ5D scores. Correlation coefficients were r = −0.25 (p = 0.011) and r = −0.47 (p < 0.0001) for HADS and EQ5D scores, respectively. Grip strength was a useful evaluation that also reflected the mental status and QOL.

Transcription activator-like effector nuclease (TALEN)-mediated genome editing is a powerful technique for analyzing gene functions in various cells and organisms. At target loci, TALENs can not only introduce short insertions and deletions, but also yield large deletions through the use of two TALEN pairs. Here, we report easy and efficient methods for enrichment of cells with TALEN-induced mutations and large deletions. First, we established the fluorescence-activated sorting of TALEN-induced deletions (FAST-id) system that enabled fluorescence-activated cell sorting-mediated enrichment of cells with TALEN-induced mutations. In the FAST-id system, either EGFP or mCherry and TALENs were co-expressed. Using dual fluorescence selection, both left and right TALEN-expressing cells were easily concentrated, resulting in enrichment of TALEN-mediated mutated cells. Next, to apply the FAST-id system to enrichment of cells with large deletions, we developed the fast unification of separate endonucleases (FUSE) method for assembly of two TALENs into a single expression vector. Using the FUSE method, we easily obtained a TALEN pair-expressing plasmid driven by a single promoter. By combining the FAST-id system and FUSE method, cells with large deletions were efficiently enriched. To the best of our knowledge, this is the first report of enrichment of cells with TALEN-induced large deletions.