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 gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal 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.

中文翻译：

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通过 dCas9-NG 介导的 CRISPR 干扰有效阻断微生物转录起始。

CRISPR 干扰 (CRISPRi) 已被开发为一种转录控制工具，通过使 Cas9 核酸酶的 DNA 切割能力失活以产生 dCas9（失活的 Cas9），并使 dCas9 能够特异性结合目标 DNA 序列。由于原型间隔区相邻基序 (PAM) (5'-NGG) 依赖于结合目标 DNA，CRISPR/Cas9 技术在设计目标特异性单向导 RNA (sgRNA) 方面存在局限性。据报道，通过Cas9的蛋白质工程去除了对PAM最后一个碱基G的依赖，构建了将5'-NG识别为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，并在大肠杆菌中的gal启动子中评估其调节转录的能力. 细胞生长速率、D-半乳糖消耗速率和gal转录物的分析证实，dCas9-NG 可以通过识别具有 5'-NGG、NGA、NGC、NGT 和 NAG 的 PAM 的 DNA 靶标来完全抑制启动子。我们的研究显示了 dCas9-NG 的可能 PAM 序列，并提供了有关用于调节基因表达和细胞代谢的目标特异性 sgRNA 设计的信息。