The CRISPR/Cas9 system, which was discovered recently, utilizes nucleases targeted by sequence complementarity and is originally intended to protect bacteria from foreign genetic elements. The system provided a convenient tool for manipulating the genomes of living cells. The CRISPR/Cas9 genomic editing technology moved beyond the laboratory and already found application in biotechnology and agriculture. However, off-target activity of the CRISPR/Cas9 system can cause oncogenic mutations and thus limits its use for genome editing in human cells for medical purposes. Many studies are therefore aimed at developing variants of the CRISPR/Cas9 system with improved accuracy. The review considers the mechanisms of precise and erroneous actions of Cas9 RNA-guided nuclease, natural and artificial variants of RNA-targeted nucleases, possibilities to modulate their specificity through guide RNA modifications, and other approaches to increasing the accuracy of the CRISPR/Cas9 system in genome editing.
Keywords: CRISPR/Cas9; Cas9 protein; enzyme specificity; genome editing; mutations; off-target effects; protein engineering.