A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9.

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

Ultrasound diagnosis of juvenile forearm fractures.

PURPOSE: Metaphyseal forearm fractures are a common pathology in any emergency department. The standard diagnostic procedure is an X-ray of the forearm and wrist. Former studies have shown that these fractures can be visualized by ultrasound. The intention of this study was to evaluate the safety and reliability of the ultrasound diagnostic procedure in comparison with X-ray diagnosis. METHODS: Patients aged 0-12 years with tentative diagnosis of forearm fracture in a physical examination were then examined, from six positions, with a 7.5-MHz linear array transducer. The diagnosis and the recommended treatment were noted, after which standard X-rays were taken. Finally, differences between diagnoses, the extent and direction of the deformity, and the treatment recommended after both diagnostic procedures were analysed. RESULTS: From January 2007 to May 2008, 93 patients were examined. We found 77 fractures in 64 patients (48 radius, 2 physeal fractures of the radius, 1 ulna, and 13 radius and ulna). The sensitivity of ultrasound in diagnosing forearm fractures was 94%, and the specificity was 99%. Difference between the means of the deformities were 1.6° (radius anterior-posterior direction), 1.6° (radius medial-lateral direction), 0.2° (ulna anterior-posterior direction), and 0.2° (ulna medial-lateral direction). CONCLUSION: Ultrasound seems to be a valuable and safe alternative to X-ray diagnosis.