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.

• 堿基編輯器（Base Editor）： 該質(zhì)粒攜帶一個(gè)融合蛋白，由鏈霉菌密碼子優(yōu)化的 rAPOBEC1（胞苷脫氨酶）、nCas9 (D10A) 和 UGI（尿嘧啶糖基化酶抑制劑）組成。

• 多重編輯機(jī)制（Multiplexing）： 與標(biāo)準(zhǔn)版相比，其名稱中的 "mc" 代表 "multiplexing-compatible"（兼容多重化）。它利用來(lái)自銅綠假單胞菌的 Csy4 核糖核酸內(nèi)切酶來(lái)處理多順?lè)醋?sgRNA 轉(zhuǎn)錄本，從而實(shí)現(xiàn)在單個(gè)實(shí)驗(yàn)中同時(shí)對(duì)多個(gè)基因組靶點(diǎn)進(jìn)行編輯。

• 編輯效果： 主要介導(dǎo) C 到 T（或 G 到 A） 的堿基轉(zhuǎn)換。在鏈霉菌中，這常被用于通過(guò)引入終止密碼子（STOP-BEST）來(lái)失活基因。

• 骨架載體： 基于 pSG5 復(fù)制子，這是一種溫度敏感型的大腸桿菌-鏈霉菌穿梭質(zhì)粒。

• 啟動(dòng)子：

  • 堿基編輯模塊： 由可誘導(dǎo)的 PtipA 啟動(dòng)子驅(qū)動(dòng)（受硫鏈絲菌素誘導(dǎo)）。

  • sgRNA 表達(dá)： 通常由 PkasO* 或 PermE* 等強(qiáng)啟動(dòng)子驅(qū)動(dòng)。

  
  

• 抗性標(biāo)記： 具有 阿普拉霉素（Apramycin） 抗性。

• 培養(yǎng)條件： 在大腸桿菌中于 37°C 培養(yǎng)；在鏈霉菌中通常在 30°C 培養(yǎng)。由于其溫度敏感性，可通過(guò)提高溫度至 37°C-39°C 輕松消除（curing）質(zhì)粒。

該系統(tǒng)廣泛應(yīng)用于鏈霉菌的代謝工程和功能基因組學(xué)研究。它能高效地在多個(gè)位位點(diǎn)同時(shí)引入點(diǎn)突變或終止密碼子，特別適用于復(fù)雜的次級(jí)代謝產(chǎn)物合成基因簇（BGC）的改造。

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