Background: RNA-guided endonuclease as a versatile genome editing technology opened
new windows in various fields of biology. The simplicity of this revolutionary technique provides a
promising future for its application in a broad range of approaches from functional annotation of genes
to diseases, to genetic manipulation and gene therapy. Besides the site-specific activity of Cas9 endonuclease,
the unintended cleavage known as off-target effect is still a major challenge for this genome
Methods: Various strategies have been developed to resolve this bottleneck including development of
new softwares for designing optimized guide RNA (gRNA), engineering Cas9 enzyme, improvement
in off-target detection assays, etc.
Results: This review dedicated to discuss on methods that have been used for optimizing Cas9, specificity
with the aim of improving this technology for therapeutic applications.
Conclusion: In addition, the applications and novel breakthroughs in the field of CRISPR technology
will be described.