ABSTRACT
Since the addition of the CRISPR/Cas9 technology to the genetic engineering toolbox, the problems of low efficiency and off-target effects hamper its widespread use in all fields of life sciences. Furthermore, essential gene knockout usually results in failure and it is often not obvious whether the gene of interest is an essential one. Here, we report on a new strategy to improve the CRISPR/Cas9 genome editing, which is based on the idea that editing efficiency is tightly linked to how essential the gene to be modified is. The more essential the gene, the less the efficiency of the editing and the larger the number of off-targets, due to the survivorship bias. Considering this, we generated deletions of three essential genes in Drosophila: trf2, top2, and mep-1, using fly strains with previous target gene overexpression ("pre-rescued" genetic background).
ABSTRACT
The DNA double helix provides a simple and elegant way to store and copy genetic information. However, the processes requiring the DNA helix strands separation, such as transcription and replication, induce a topological side-effect - supercoiling of the molecule. Topoisomerases comprise a specific group of enzymes that disentangle the topological challenges associated with DNA supercoiling. They relax DNA supercoils and resolve catenanes and knots. Here, we review the catalytic cycles, evolution, diversity, and functional roles of type II topoisomerases in organisms from all domains of life, as well as viruses and other mobile genetic elements.