Targeted genome editing using engineered nucleases such as ZFNs and TALENs has been rapidly replaced by the CRISPR/Cas9 (clustered, regulatory interspaced, short palindromic/ CRISPR-associated nuclease) system. CRISPR/Cas9 technology represents a significant improvement enabling a new level of targeting, efficiency and simplicity. Gene editing mediated by CRISPR/Cas9 has been recently used not only in bacteria but in many eukaryotic cells and organisms, from yeasts to mammals. Other modifications of the CRISPR-Cas9 system have been used to introduce heterologous domains to regulate gene expressions or label specific loci in various cell types. The review focuses not only on native CRISPR/Cas systems which evolved in prokaryotes as an endogenous adaptive defense mechanism against foreign DNA attacks, but also on the CRISPR/Cas9 adoption as a powerful tool for site-specific gene modifications in fungi, plants and mammals.