Improving CRISPR-Cas9 On-Target Specificity

The CRISPR/Cas9 has revolutionized the field of molecular biology, medical genetics and medicine. The technology is robust, facile and simple to achieve genome targeting in cells and organisms. However, to propagate these nucleases for therapeutic application, the on-target specificity is of paramount importance. Although the binding and cleavage of off-target sites by Cas9 is an issue of concern, however, the specificity of CRISPR technology is greatly improved in current research employing the use of engineer nucleases, improved gRNA selection, novel Cas9 orthologues and the advancement in methods to detect and screen offtarget sites and its effects. Here we summarize the advances in this state-of-the-art technology that will equip the genome editing tools to be applied in clinical research. The researcher should optimize these methods with emphasis to achieving perfection in the specificity.