The CRISPR/Cas9 system was initially described as an element of archeal and bacterial adaptive immunity, but has recently gained much attention in genetic engineering due to its outstanding ability to be programmed to target any genomic loci through a short 20-nucleotide region of single guide RNA (sgRNA). This review deals with some modern applications of the CRISPR/Cas9 system. First, we describe the basic mechanism of the CRISPR/Cas9 DNA recognition and binding with a particular focus on its off-target activity. The CRISPR/Cas9 off-target activity refers to nonspecific recognition of genomic sites that have partial homology with sgRNA. Such activity occasionally results in unwanted mutations throughout the genome. We also note some recent method improvements for enhancing Cas9 specificity or adding new functions to the system. Since the active use of CRISPR/Cas9 is mostly driven by its remarkable potential for gene therapy and genome engineering, the latest CRISPR/Cas9 applications in these areas are also discussed in our review. In particular, the CRISPR/Cas9 system was recently used to control the human immunodeficiency virus (HIV) infection and to repair genetic abnormalities, such as Duchenne muscular dystrophy or retinitis pigmentosa, both in cell cultures and rodent models. The programmable nature of CRISPR/Cas9 facilitates the creation of transgenic organisms through site-specific gene mutations, knock-ins or large chromosomal rearrangements (deletions, inversions and duplications). CRISPR/Cas9 was also found to be especially useful in pronuclear microinjections of farm animals, which has a strong impact on biotechnology. In addition, we reviewed CRISPR/Cas9-based genetic screens, which provide highly efficient identification of new genes and pathways involved in many biological processes. Finally, we discuss the use of gene drivers based on CRISPR/Cas9, which represent a powerful tool for the modification of ecosystems in the nearest future.