CRISPR/Cas9 is a bacterial immune system against invading viruses. Because altering the sgRNA sequence, which binds to the target in the genome is so facile, CRISPR/Cas9 has become a method of choice in the fields such as genetic engineering, microbial biotechnology or synthetic biology. Furthermore, the system has a great potential in clinical applications for correcting disease-causing genes in somatic cells. The potential of CRISPR/Cas9 extends further than gene editing via NHEJ or HDR. Applications of deactivated Cas9 protein with a fused effector domain have seen a surge in popularity in recent years. With them, it is possible to alter gene regulation, cause epigenetic remodeling or visualize certain loci of interest. Although there is a wide range of potential uses of the system, it is currently coping with some limitations. Such limitations are off-target mutations or problems with delivery. Recently, new solutions have emerged for those challenges, such as engineered Cas9 proteins, modifications in sgRNA and some new systems for higher specificity. It is crucial to determine potential off target sites in both in silico and in vivo or in vitro systems.
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