Animal Technology Research Center, Division of Animal Technology, Agricultural Technology Research Institute, No. 52, Kedong 2nd Rd., Zhunan Township, Miaoli County 35053, Taiwan.
Int J Mol Sci. 2021 Sep 13;22(18):9872. doi: 10.3390/ijms22189872.
Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (HA) and RuvC nuclease activity null mutant (DA) were identified. In accompaniment with the sgRNA, Cas9, Cas9(DA), Cas9(HA), and Cas9(DA, HA) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.
从理论上讲，能够区分长度等于或超过 16 个碱基的 DNA 序列的 DNA 序列特异性识别蛋白可能是哺乳动物基因组所特有的。已经开发并广泛应用了长序列特异性核酸酶，如天然存在的归巢内切核酸酶和人工设计的 ZFN、TALEN 和 Cas9-sgRNA，用于基因组编辑。与其他通过蛋白部分自身识别 DNA 靶位点的对应物不同，Cas9 使用单链向导 RNA（sgRNA）作为 DNA 靶标识别的模板。由于针对靶位点设计和合成 sgRNA 非常简单，Cas9-sgRNA 已成为当前基因组编辑的最常用工具。此外，Cas9-sgRNA 的 RNA 引导的 DNA 识别活性独立于其 HNH 结构域对互补链的核酸酶活性和 RuvC 结构域对非互补链的核酸酶活性，并且鉴定了 HNH 核酸酶活性缺失突变体（HA）和 RuvC 核酸酶活性缺失突变体（DA）。与 sgRNA 一起，Cas9、Cas9(DA)、Cas9(HA)和 Cas9(DA、HA) 可分别用于在靶位点实现双链断裂、互补链断裂、非互补链断裂和无断裂。基于这些独特的特性，可以在靶位点内或周围引入许多工程酶活性，如 DNA 甲基化、组蛋白甲基化、组蛋白乙酰化、胞嘧啶脱氨酶、腺嘌呤脱氨酶和引物指导的突变。为了防止 Cas9 衍生物的持续表达导致脱靶，可以开发许多瞬时表达方法，包括 Cas9-sgRNA 核糖核蛋白的直接递送。在体内应用中，生物安全性是必不可少的；已经设计了 Cas9-sgRNA 包装到病毒样颗粒或细胞外囊泡中，并报告了一些体内治疗试验。
