Efforts to improve the efficiency and specificity of CRISPR-Cas9 techniques

The CRISPR-Cas9 technique derived from bacterial immune system has been used in the majority of genome engineering applications due to its advantages of low cost and flexibility. In order to achieve efficient modifications to target genome in different cell types by CRISPR-Cas9 genome editing, three basic components are required, including 20nt sequence of sgRNA that could recognize desired DNA sequences, Cas9 protein for introducing double-strand breaks and the NGG PAM sequence for the binding between Cas9 proteins and the target genomic sites. However, the CRISPR-Cas9 technique today are facing some difficulties in adoption of further genome applications, especially those requiring high precision modifications. This is mainly because of the off-target effects, in which the spCas9 nuclease cleaves unwanted genomic sequences. To address this, a number of strategies have been developed. In this article, we mainly reviewed the factors in determining the activity of Cas9 proteins and concluded the approaches to reduce the off-target effects.