The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters.

BackgroundAustralia’s health disparity, combined with evolving technologies, has evoked increasing interest and funding in health services that could address inequities. One such emerging service is tele-medicine.ObjectiveThe purpose of this report is to discuss and evaluate the current literature regarding patient and practitioner satisfaction with tele-medicine, and more specifically tele-dermatology.MethodsWe searched for literature relevant to tele-dermatology use among Australia’s indigenous population. We synthesized the literature in our report and identified elements of tele-dermatology not yet researched.ResultsMost significantly, all available research is currently based on descriptive studies and there is no validated tool to assess the efficacy of tele-dermatology.LimitationsNo published research currently exists on the use of tele-dermatology among Australia’s indigenous population.ConclusionA review of the literature shows that tele-dermatology is considered a valuable service, particularly to patients living in rural areas who might not otherwise have access to specialist care.

A novel mechanism is proposed for ring formation in the biosynthetic pathway to thiotetronate antibiotics thiolactomycin and Tü 3010.