Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0653-x) contains supplementary material, which is available to authorized users.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

Maladaptation to stress is a critical risk factor in stress-related disorders, such as major depression and post-traumatic stress disorder (PTSD). Dopamine signaling in the nucleus accumbens (NAc) has been shown to modulate behavior by reinforcing learning and evading aversive stimuli, which are important for the survival of animals under environmental challenges such as stress. However, the mechanisms through which dopaminergic transmission responds to stressful events and subsequently regulates its downstream neuronal activity during stress remain unknown. To investigate how dopamine signaling modulates stress-coping behavior, we measured the subsecond fluctuation of extracellular dopamine concentration and pH using fast scanning cyclic voltammetry (FSCV) in the NAc, a postsynaptic target of midbrain dopaminergic neurons, in male mice engaged in a tail suspension test (TST). The results revealed a transient decrease in dopamine concentration and an increase in pH levels when the animals changed behaviors, from being immobile to struggling. Interestingly, optogenetic inhibition of dopamine release in NAc, potentiated the struggling behavior in animals under the TST. We then addressed the causal relationship of such a dopaminergic transmission with behavioral alterations by knocking out both the dopamine receptors, i.e., D1 and D2, in the NAc using viral vector-mediated genome editing. Behavioral analyses revealed that male D1 knock-out mice showed significantly more struggling bouts and longer struggling durations during the TST, while male D2 knock-out mice did not. Our results therefore indicate that D1 dopaminergic signaling in the NAc plays a pivotal role in the modulation of stress-coping behaviors in animals under tail suspension stress.

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BackgroundFusion genes have been recognized to play key roles in oncogenesis. Though, many techniques have been developed for genome-wide analysis of fusion genes, a more efficient method is desired.ResultsWe introduced a new method of detecting the novel fusion gene by using GeneChip Exon Array that enables exon expression analysis on a whole-genome scale and TAIL-PCR. To screen genes with abnormal exon expression profiles, we developed computational program, and confirmed that the program was able to search the fusion partner gene using Exon Array data of T-cell acute lymphocytic leukemia (T-ALL) cell lines. It was reported that the T-ALL cell lines, ALL-SIL, BE13 and LOUCY, harbored the fusion gene NUP214-ABL1, NUP214-ABL1 and SET-NUP214, respectively. The program extracted the candidate genes with abnormal exon expression profiles: 1 gene in ALL-SIL, 1 gene in BE13, and 2 genes in LOUCY. The known fusion partner gene NUP214 was included in the genes in ALL-SIL and LOUCY. Thus, we applied the proposed program to the detection of fusion partner genes in other tumors. To discover novel fusion genes, we examined 24 breast cancer cell lines and 20 pancreatic cancer cell lines by using the program. As a result, 20 and 23 candidate genes were obtained for the breast and pancreatic cancer cell lines respectively, and seven genes were selected as the final candidate gene based on information of the EST data base, comparison with normal cell samples and visual inspection of Exon expression profile. Finding of fusion partners for the final candidate genes was tried by TAIL-PCR, and three novel fusion genes were identified.ConclusionsThe usefulness of our detection method was confirmed. Using this method for more samples, it is thought that fusion genes can be identified.