Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea.
J Microbiol Biotechnol. 2020 Dec 28;30(12):1919-1926. doi: 10.4014/jmb.2008.08058.
CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the promoter in . Analysis of cell growth rate, D-galactose consumption rate, and transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.
CRISPR 干扰 (CRISPRi) 通过使 Cas9 核酸酶的 DNA 切割能力失活来产生 dCas9（失活 Cas9），从而被开发为转录控制工具，并使 dCas9 具有特异性结合靶 DNA 序列的能力。由于依赖于前间隔基序 (PAM) (5'-NGG) 来结合靶 DNA，CRISPR/Cas9 技术在设计靶特异性单指导 RNA (sgRNA) 方面存在局限性。据报道，通过 Cas9 的蛋白质工程去除对 PAM 最后一个碱基 G 的依赖性，构建了识别 5'-NG 作为 PAM 序列的 Cas9-NG。在这项研究中，通过在 Cas9-NG 中引入两个活性位点突变来设计 dCas9-NG 蛋白，并在. 中评估其在启动子中调节转录的能力。细胞生长速率、D-半乳糖消耗速率和 转录本的分析证实，dCas9-NG 可以通过识别具有 5'-NGG、NGA、NGC、NGT 和 NAG PAM 的 DNA 靶标完全抑制启动子。我们的研究显示了 dCas9-NG 的可能 PAM 序列，并提供了有关针对特定 sgRNA 设计的信息，用于调节基因表达和细胞代谢。
