Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli.

CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, we show that even single-stranded oligo nucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, and in A. oryzae indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, we demonstrate that by using single-stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions at efficiencies approaching 100%. The efficiency of the system invites for multiplexing and we have designed a vector system with the capacity of delivering Cas9 and multiple sgRNAs based on polymerase III promoters and tRNA spacers. We show that it is possible to introduce two point mutations and one gene insertion in one transformation experiment with a very high efficiency. Our system is compatible with future high-throughput gene-editing experiments.

Post-mastectomy radiotherapy in Denmark: from 2D to 3D treatment planning guidelines of The Danish Breast Cancer Cooperative Group.

This paper describes the procedure of changing from 2D to 3D treatment planning guidelines for post-mastectomy radiotherapy in Denmark. The aim of introducing 3D planning for post-mastectomy radiotherapy was to optimize the target coverage and minimize the dose to the normal tissues. Initially, it was investigated whether it was possible to find a treatment technique alternative to the one recommended by the Danish Breast Cancer Cooperative Group (DBCG). A dosimetric comparison of a combined photon/electron 3-field technique (3F) and a partial wide tangent technique (PWT) was carried out on individual planning CT-scans from seven patients selected to represent a wide range of sizes and shapes of chest walls. The heart dose was lower for PWT than for 3F, however, for both techniques the dose was within the accepted constraints. The lung dose was higher but acceptable for six of the seven patients with PWT. The dose to the internal mammary nodes (IMN) was not satisfactory for five of the seven patients for 3F, whereas only two of the seven patients had a minimum dose lower than 95% of the prescribed dose with PWT. Finally, the dose to the contralateral breast was increased when using PWT compared to 3F. It was concluded that PWT was an appropriate choice of technique for future radiation treatment of post-mastectomy patients. A working group was formed and guidelines for 3D planning were developed during a series of workshops where radiation oncologists and physicists from all radiotherapy centres participated. This work also included a definition of the tissue structures needed to be outlined on the planning CT-scan. The work was initiated in 2003 and the guidelines were approved by the DBCG Radiotherapy Committee in 2006. The first of January 2007 the 3D guidelines had been fully implemented in five of the seven radiotherapy centres.