Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish.

Gene modifications in animal models have been greatly facilitated through the application of targeted genome editing tools. The prokaryotic CRISPR/Cas9 type II genome editing system has recently been applied in cell lines and vertebrates. However, we still have very limited information about the efficiency of mutagenesis, germline transmission rates and off-target effects in genomes of model organisms. We now demonstrate that CRISPR/Cas9 mutagenesis in zebrafish is highly efficient, reaching up to 86.0%, and is heritable. The efficiency of the CRISPR/Cas9 system further facilitated the targeted knock-in of a protein tag provided by a donor oligonucleotide with knock-in efficiencies of 3.5-15.6%. Mutation rates at potential off-target sites are only 1.1-2.5%, demonstrating the specificity of the CRISPR/Cas9 system. The ease and efficiency of the CRISPR/Cas9 system with limited off-target effects make it a powerful genome engineering tool for in vivo studies.

Genetic predisposition for high stress reactivity amplifies effects of early-life adversity.

A dysregulation of the hypothalamus-pituitary-adrenocortical (HPA) axis and the experience of early-life adversity are both well-established risk factors for the development of affective disorders, such as major depression. However, little is known about the interaction of these two factors in shaping endophenotypes of the disease. Here, we studied the gene-environment interaction of a genetic predisposition for HPA axis dysregulation with early-life stress (ELS), assessing the short-, as well as the long-lasting consequences on emotional behavior, neuroendocrine functions and gene expression profiles. Three mouse lines, selectively bred for either high (HR), intermediate (IR), or low (LR) HPA axis reactivity, were exposed to one week of ELS using the limited nesting and bedding material paradigm. Measurements collected during or shortly after the ELS period showed that, regardless of genetic background, ELS exposure led to impaired weight gain and altered the animals' coping behavior under stressful conditions. However, only HR mice additionally showed significant changes in neuroendocrine stress responsiveness at a young age. Accordingly, adult HR mice also showed lasting consequences of ELS, including hyperactive stress-coping, HPA axis hyperreactivity, and gene expression changes in the Crh system, as well as downregulation of Fkbp5 in relevant brain regions. We suggest that the genetic predisposition for high stress reactivity interacts with ELS exposure by disturbing the suppression of corticosterone release during a critical period of brain development, thus exerting lasting programming effects on the HPA axis, presumably via epigenetic mechanisms. In concert, these changes lead to the emergence of important endophenotypes associated with affective disorders.