CRISPR基因编辑的脱靶效应应对策略综述

Strategies against Off-target Effects in CRISPR/Cas9-mediated Genome Editing

CRISPR/Cas9基因编辑系统是第三代基因编辑技术,在生物医学领域有着广泛的应用。该系统由sgRNA和Cas9蛋白组成,sgRNA与Cas9结合并引导Cas9到达DNA靶点,Cas9作为核酸内切酶对靶序列进行切割,形成DNA双链断裂(double strand break, DSB)。DSB通过两种机制——非同源末端连接(nonhomologous end joining, NHEJ)和同源重组介导的修复(homology-directed repair, HDR)——进行修复,实现特定基因的敲除或插入。与锌指核酸酶(ZFNs)和转录激活因子样效应物核酸酶(TALENs)等基因编辑工具相比,CRISPR/Cas9在简便性、专一性等方面有很大优势,但脱靶效应一直是它在实际应用中面临的一个难题。目前已有许多针对脱靶效应的研究,科学家们已发现一些影响靶点专一性的因素,如sgRNA种子序列、PAM序列、Cas9蛋白、DSB修复通路等,并据此研发出降低脱靶效应的可行性策略。本综述就CRISPR/Cas9系统的作用原理、潜在的脱靶危害和降低脱靶效应的策略研究进行阐述。

CRISPR/Cas9 genome-editing system has been regarded as the third generation genome-editing technology which is widely used in biomedicine. The system consists of sgRNAs and Cas9 proteins. In this system,sgRNA binds to Cas9 and directs it to target DNA. Cas9 cleaves target DNA as an endonuclease, forming DNA double strand breaks(DSB). DSB can be repaired through two mechanisms—nonhomologous end joining(NHEJ)and homology-directed repair(HDR), to achieve knockout or insertion of a specific gene. Compared with other genome-editing tools, such as zinc finger nucleases(ZFNs) and transcription activator-like effector nucleases(TALENs),CRISPR/Cas9 has great advantages in simplicity and specificity. However, off-target effect remains one of the most difficult challenges in the application of CRISPR/Cas9. So far, there have been many studies on off-target effects. Some factors influencing target specificity have been found, such as sgRNA seed sequence, PAM sequence, Cas9 proteins, DNA repair pathways, etc. Some strategies to reduce off-target effects have been developed accordingly. Here, we review the genome editing mechanism of CRISPR/Cas9 system, the potential off-target hazard and the strategies to reduce off-targeting.