摘要
CRISPR/Cas9是近年来发展起来的新兴技术,其在多种生物和组织的基因组上具有快速、高效、精准的基因编辑与调控能力,这使得该技术在基础科学和合成生物学等应用科学领域均得到了极大的发展与应用。本文首先对CRISPR的历史沿革、分类及CRISPR/Cas9技术的作用机制进行简述,并结合其原理和在基因组工程中面临的脱靶率高、PAM依赖性强等限制因素总结了近年来针对Cas9蛋白和向导RNA (gRNA)进行的一系列优化与改造。接下来详细叙述了CRISPR/Cas9系统结合效应蛋白实现的多种功能,包括基因表达调控、表观基因组编辑、单碱基编辑等。基于gRNA多表达策略和Cas9多路复用策略,本文还对CRISPR/Cas9技术主要的多重应用成果进行了梳理汇总。最后探讨了CRISPR/Cas9作为高精度基因组编辑工具使用的应用前景,以及作为疗法使用时的安全性和风险控制问题。
As an emerging technology developed within recent years, CRISPR/Cas9 exhibits fast, efficient, and precise gene editing and regulation capabilities in various organisms and tissues, and these advantages make it widely used in research with fundamental sciences and applied technologies as well such as synthetic biology. This review first briefly introduces the discovery history, classification, and mechanism of CRISPR/Cas9. The system of CRISPR/Cas9usually contains a single guide RNA(gRNA) molecule for targeting a specific sequence, and a Cas9 endonuclease for catalyzing a double-strand break(DSB) in the sequence(target DNA strands). The recognition and cleavage of target DNA strictly require the presence of a protospacer adjacent motif(PAM) in the target sequence. The DSB(s) can be repaired by various DNA repair mechanisms, which allow various gene editing such as gene integration, gene replacement, and gene knockout. Due to limitations of CRISPR/Cas9, such as PAM dependence and high off-target rate, researchers have developed various fused or engineered Cas9 proteins and g RNAs that play significant roles in fulfilling various purposes. These Cas9 variants are modified for improving the performance of PAM, in particular its specificity and fidelity. Moreover, the DSBs generated by Cas9 are considered toxic to the cells, and the use of Cas9 nickase(n Cas9) or catalytically deficient Cas9(d Cas9) in CRISPR has also been developed without generating DSBs. Meanwhile, different effector proteins can be fused with Cas9/d Cas9/n Cas9 to bring about new functions and applications in gene expression regulation, epigenome editing, and single base editing. Moreover, we introduce the current studies and applications of multiple g RNA expression strategies based on the multiplex advantages of the CRISPR/Cas9 system. In general, CRISPR/Cas9 systems have gradually become standardized and revolutionized genome editing systems for almost all possible genetic manipulations. Finally, we highlight perspectives on several applicatio
作者
滕小龙
史硕博
TENG Xiaolong;SHI Shuobo(Beijing Advanced Innovation Center of Soft Matter Science and Engineering,Beijing University of Chemical Technology,Beijing 100029,China)
出处
《合成生物学》
CSCD
2023年第1期67-85,共19页
Synthetic Biology Journal
基金
国家自然科学基金(21878013)。
