Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

Background: Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. Results: Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Conclusions: Our results provide a technical platform for high-throughput knock-in

A tryptophan-rich breakfast and exposure to light with low color temperature at night improve sleep and salivary melatonin level in Japanese students

Background: Epidemiological studies in Japan have documented an association between morning type and a tryptophan-rich breakfast followed by exposure to sunlight in children. The association may be mediated by enhanced melatonin synthesis, which facilitates sleep at night. However, melatonin is inhibited by artificial light levels with high color-temperature common in Japanese homes at night. In this study, we investigated whether a combination of tryptophan-rich breakfast and light with low color-temperature at night could enhance melatonin secretion and encourage earlier sleep times Methods: The intervention included having breakfast with protein- and vitamin B6 - rich foods and exposure to sunlight after breakfast plus exposure to incandescent light (low temperature light) at night (October-November, 2010). The participants were 94 members of a university soccer club, who were divided into 3 groups for the intervention (G1: no intervention; G2: asked to have protein-rich foods such as fermented soybeans and vitamin B6-rich foods such as bananas at breakfast and sunlight exposure after breakfast; G3: the same contents as G2 and incandescent light exposure at night). Salivary melatonin was measured around 11:00 p.m. on the day before the beginning, a mid-point and on the day before the last day a mid-point and on the last day of the 1 month intervention. Results: In G3, there was a significantly positive correlation between total hours the participants spent under incandescent light at night and the frequency of feeling sleepy during the last week (p = 0.034). The salivary melatonin concentration of G3 was significantly higher than that of G1 and G2 in combined salivary samplings at the mid-point and on the day before the last day of the 1 month intervention (p = 0.018), whereas no such significant differences were shown on the day just before the start of the intervention (p = 0.63). Conclusion: The combined intervention on breakfast, morning sunlight and evening-lighting seems to be effective for students including athletes to keep higher melatonin secretion at night which seems to induce easy onset of the night sleep and higher quality of sleep

Additional file 1: of Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

The following additional data are available with the online version of this paper. Additional data file 1 contains the figures including IDA analysis of PITCh-donor, PCR screenings of mice, sequencing of non-knock-in Actb alleles, FACS and LSM analysis of human cells, Exo1 western blotting, Exo1 toxicity analysis, off-target analysis in mice, germline transmission, linear PCR donor injection, and sequence alignments of hACTB and its off-target sites, and tables of these results and a list of the oligo DNAs and RNAs used in this study. (DOCX 14447 kb