pCRISPR-mcBEST链霉菌多重胞嘧啶碱基编辑质粒载体BioVector®

BioVector® pCRISPR-mcBEST (BioVector®Plasmid #209416) is a specialized tool for multiplexed cytosine base editing in Streptomyces species.It is part of the CRISPR-BEST (Base Editing SysTem) toolkit, which allows for precise genome modifications without requiring double-strand breaks (DSBs).

Core Components & Function

• Editor Cassette: It carries a fusion protein consisting of a Streptomyces codon-optimized rAPOBEC1 (cytidine deaminase), nCas9 (D10A), and UGI (uracil glycosylase inhibitor).

• Multiplexing Mechanism: Unlike standard pCRISPR-cBEST, the "mc" (multiplexed cytosine) version utilizes the Csy4 endoribonuclease (from Pseudomonas aeruginosa) to process polycistronic sgRNA transcripts, allowing for simultaneous editing of multiple genomic targets.

• Editing Window: It facilitates C-to-T (or G-to-A) conversions within a specific window relative to the Protospacer Adjacent Motif (PAM), which is 5′-NGG-3′ for this system.

• Backbone: Based on a pSG5 replicon, making it a temperature-sensitive E. coli–Streptomyces shuttle plasmid.

• Promoters:

  • Base Editor: Driven by the inducible PtipA promoter (responsive to thiostrepton).

  • sgRNA Expression: Typically driven by promoters such as PkasO* or PermE*.

  
  

• Resistance: Confers resistance to Apramycin (25 μg/mL).

• Growth Conditions: Standard growth in E. coli (DH5alpha) is at 37°C, while growth in Streptomyces is typically conducted at 30°C due to its temperature-sensitive nature, which facilitates plasmid curing after editing.

The system is widely used for metabolic engineering and functional genomics in actinomycetes, particularly for introducing stop codons (STOP-BEST) or specific amino acid substitutions at multiple loci in a single step.

• 碱基编辑器（Base Editor）： 该质粒携带一个融合蛋白，由链霉菌密码子优化的 rAPOBEC1（胞苷脱氨酶）、nCas9 (D10A) 和 UGI（尿嘧啶糖基化酶抑制剂）组成。

• 多重编辑机制（Multiplexing）： 与标准版相比，其名称中的 "mc" 代表 "multiplexing-compatible"（兼容多重化）。它利用来自铜绿假单胞菌的 Csy4 核糖核酸内切酶来处理多顺反子 sgRNA 转录本，从而实现在单个实验中同时对多个基因组靶点进行编辑。

• 编辑效果： 主要介导 C 到 T（或 G 到 A） 的碱基转换。在链霉菌中，这常被用于通过引入终止密码子（STOP-BEST）来失活基因。

• 骨架载体： 基于 pSG5 复制子，这是一种温度敏感型的大肠杆菌-链霉菌穿梭质粒。

• 启动子：

  • 碱基编辑模块： 由可诱导的 PtipA 启动子驱动（受硫链丝菌素诱导）。

  • sgRNA 表达： 通常由 PkasO* 或 PermE* 等强启动子驱动。

  
  

• 抗性标记： 具有 阿普拉霉素（Apramycin） 抗性。

• 培养条件： 在大肠杆菌中于 37°C 培养；在链霉菌中通常在 30°C 培养。由于其温度敏感性，可通过提高温度至 37°C-39°C 轻松消除（curing）质粒。

该系统广泛应用于链霉菌的代谢工程和功能基因组学研究。它能高效地在多个位位点同时引入点突变或终止密码子，特别适用于复杂的次级代谢产物合成基因簇（BGC）的改造。

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