Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

Cardiorespiratory fitness, sleep, and physiological responses to stress in women.

Epidemiological studies suggest that cardiorespiratory fitness (CRfitness) is associated with reduced risk of depression and anxiety in women, however, the mechanisms by which CRfitness may be protective against the development of these disorders are less clear. Because sleep problems are associated with both a higher risk for mental illness and altered physiological responses to stress, this study investigated whether sleep quality might influence the relationship between CRfitness and physiological stress responses in women. Thirty healthy women (18-45 y) who were medication-free, with regular menstrual cycles completed: (1) enrolment visit [including the assessment of CRfitness via maximal oxygen consumption during exercise]; (2) one-week sleep monitoring period including subjective (daily sleep diaries) and objective (wrist actigraphy) sleep measures; and (3) psychosocial stressor protocol (the Trier Social Stress Test; TSST) for the collection of heart rate (HR), blood pressure (BP), and salivary cortisol stress responses. Higher CRfitness was associated with reduced wake after sleep onset (WASO) duration (r = -.38, p = 0.04), higher self-reported sleep quality (higher scores reflect poorer sleep quality; r = -.37, p = 0.05), and lower HR (r = -.43, p = 0.02) during the stressor. Higher sleep quality was associated with a lower HR during the stressor (r = .44, p = 0.01). Increased WASO duration and WASO number were associated with blunted cortisol output during the stressor (r = -.44, p = 0.02, and r = -.46, p = 0.02, respectively). Results suggest that, in women, CRfitness may be protective against the deleterious effects of stress via improved sleep quality.