调控CRISPR-Cas9系统用于基因编辑的研究进展

Recent Progress in Regulating CRISPR-Cas9 System for Gene Editing

基于操作简便、成本低、快捷高效的优势,规律间隔成簇短回文重复序列及其相关核酸酶(CRISPR-Cas9)系统在基因编辑的基础研究和临床医学方面起到了极为重要的推动作用.在时空维度上调控CRISPR-Cas9发挥功能,对于减少CRISPR-Cas9系统的脱靶效应,提高基因编辑的特异性具有重要意义.本综述介绍了近年来利用化学分子以及光调控CRISPR-Cas9系统发挥功能,进而调控基因编辑的研究进展,并探讨了CRISPR-Cas9调控的前景和面临的挑战.

Clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system is an adaptive immune system used by many bacteria and archaea to defend the invasion of exogenous nucleic acids. CRISPR-Cas system in different species of archaea and bacteria has different components and working mechanisms. Depending on the numbers of effector proteins, CRISPR-Cas systems can be classified into two major types. CRISPR-Cas9, which is composed of Cas9 nuclease and sgRNA, belongs to class II CRISPR-Cas system and can be used as a powerful genome editing tool. It can target and cleave the DNA sequence which contains protospacer adjacent motif(PAM, 5’-NGG-3’) sequence. The DNA double-strand breaks(DSBs) can be repaired by homology-directed repair(HDR) or nonhomologous end joining(NHEJ) mechanism. Insertions or deletions(indels) can be introduced at targeted loci in the DSBs repair process. Due to its convenience, low cost and high efficiency, CRISPR-Cas9 has played an important role in promoting the development of gene editing in basic research and clinical medicine. However, off-target effect of CRISPR-Cas9 should not be neglected. The CRISPR-Cas9 is able to cleave the target DNA even when the sgRNA imperfectly matches with the target DNA, leading to the unwanted indels at nontargeted DNA loci, which limits the further application of genome editing, especially for the treatment of genetic diseases. Therefore, it is significant to reduce the off-target cleavage effect of CRISPR-Cas9. Many efforts have been devoted to realize the reduced off-target effect of CRISPR-Cas9. Among these methods, regulating the function of CRISPR-Cas9 at spatiotemporal dimension is a potential strategy to reduce the off-target effect of CRISPR-Cas9 system and improve the specificity of gene editing. In this review, we summarized the research advances in regulating the function of CRISPR-Cas9 and discussed the prospects and challenges of CRISPR-Cas9 regulation.