College of Health and Life Sciences, Division of Biological and Biomedical Sciences, Hamad Bin Khalifa University, Education City, Qatar Foundation, Doha P.O.Box 34110, Qatar.
Cells. 2020 Nov 21;9(11):2518. doi: 10.3390/cells9112518.
The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR-FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.
经鉴定，稳健的簇状规律间隔短回文重复序列（CRISPR）相关内切酶（Cas9）系统基因编辑工具为广泛的潜在治疗应用打开了大门，这些应用受到更复杂工具的限制，包括锌指核酸酶（ZFNs）和转录激活因子样效应核酸酶（TALENs）。然而，CRISPR 系统脱靶活性的高频率仍然限制了其应用，因此，用于高度特异性 CRISPR/Cas9 介导的基因组编辑的先进策略正在不断开发中，包括 CRISPR-FokI 失活 Cas9（fdCas9）。fdCas9 系统源自将 FokI 内切酶催化结构域与无活性 Cas9 蛋白连接，并需要一对 sgRNA，这些 sgRNA 结合在原间隔区邻近基序（PAM）的方向上的 DNA 的有义链和反义链上，在靶位点周围具有定义的间隔序列范围。FokI 结构域的二聚化产生 DNA 双链断裂，激活 DNA 修复机制，导致基因组编辑。到目前为止，与其他平台相比，所有在人类细胞中设计的工程化 fdCas9 变体都显示出有希望的基因编辑活性。在此，我们综述了所有已发表的 fdCas9 变体的优势及其在基因组工程中的当前应用。
