The Double Nickase Plasmid features a U6 promoter for sgRNA expression, a 20 nt targeting sequence, and a gRNA scaffold to guide Cas9n. It includes a CBh promoter for Cas9n (D10A) and puromycin resistance, GFP for transfection verification, and nuclear localization signals (NLS). The 2A peptide allows co-expression of Cas9n and Puro from a single promoter, enabling precise genome editing with reduced off-target effects.
The Double Nickase Plasmid features a U6 promoter for sgRNA expression, a 20 nt targeting sequence, and a gRNA scaffold to guide Cas9n. It includes a CBh promoter for Cas9n (D10A) and puromycin resistance, GFP for transfection verification, and nuclear localization signals (NLS). The 2A peptide allows co-expression of Cas9n and Puro from a single promoter, enabling precise genome editing with reduced off-target effects.
Cas9n Nickase gRNA Plasmid Targeting: Dual gRNA plasmids create single-strand nicks at precise DNA sequences for efficient genome editing using Cas9n Nickase.
This image illustrates the Cas9n Nickase mechanism used for precise genome editing. Two plasmids (Plasmid 1 and Plasmid 2) are shown, each containing a targeted DNA sequence. The system utilizes single-guide RNAs (sgRNA) to direct Cas9n Nickase to specific genomic locations, represented by the blue and pink DNA strands. The sgRNA scaffold aids in guiding Cas9n to the 20 nucleotide (nt) target sequence on the DNA. Cas9n makes single-strand cuts at NCC and NGG sites, enabling precise gene modifications without creating double-strand breaks.
The Double Nickase Plasmid features a U6 promoter for sgRNA expression, a 20 nt targeting sequence, and a gRNA scaffold to guide Cas9n. It includes a CBh promoter for Cas9n (D10A) and puromycin resistance, GFP for transfection verification, and nuclear localization signals (NLS). The 2A peptide allows co-expression of Cas9n and Puro from a single promoter, enabling precise genome editing with reduced off-target effects.
BRF1 编码 TFIIIB90-1,它是 RNA 聚合酶 III(Pol III)转录因子 TFIIIB 的核心亚基之一,并与 TBP 和 BDP1 协同作用,将 Pol III 定位到 tRNA 基因、5S rRNA 以及其他小型非编码 RNA 位点的启动子上。通过控制 Pol III 的转录起始,TFIIIB90-1 影响核糖体生物发生、翻译能力和细胞生长程序,从而将营养感知与应激响应型转录调控同细胞增殖联系起来。在多种背景下,Pol III 产出失衡以及 TFIIIB 组分活性异常与致癌转化和蛋白质稳态改变有关,使 BRF1 成为解析生物合成通路转录控制的一个有用节点。对 BRF1 的功能研究通常与 Pol III 启动子处的染色质调控、细胞周期进程以及调节 Pol III 转录通量的代谢信号传导相交织。
TFIIIB90-1 双切酶质粒(h)由一对匹配的质粒组成,专为在 human 细胞系中对 BRF1 位点进行高特异性编辑而设计。每个质粒分别表达Cas9 D10A切口酶和针对BRF1内不同DNA链的独特sgRNA。当这两种切口酶被引导至相邻但位于DNA链相反侧的位点时,会产生错位的单链切口,从而共同形成错位双链断裂,这需要两个引导RNA在靶位点上协同发挥作用。由此产生的DNA断裂通过内源性细胞修复途径(最常见的是非同源末端连接(NHEJ))得到修复,从而导致插入或缺失,进而破坏BRF1的功能。通过要求双sgRNA在靶位点结合,双切口方法提高了编辑特异性,并为需要对靶向精度进行额外控制的应用提供了互补的CRISPR策略。