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.
SDHC 编码琥珀酸脱氢酶复合体的膜锚定亚基 C,是线粒体复合体 II 的关键组成部分。复合体 II 通过促进琥珀酸氧化并将电子传递给泛醌,把三羧酸(TCA)循环与电子传递链连接起来。作为线粒体内膜锚定结构的一部分,SDHC 有助于稳定复合体 II 的组装并保障高效的氧化磷酸化,从而影响细胞的氧化还原平衡及活性氧(ROS)的处理。SDHC 功能受损会削弱线粒体呼吸并导致琥珀酸积累,继而通过改变 α-酮戊二酸(α-KG)依赖性双加氧酶的活性,对代谢信号传导和表观遗传调控产生下游影响。SDHC 的改变与线粒体功能障碍相关,并被认为参与遗传性副神经节瘤/嗜铬细胞瘤的发生机制,因此是研究代谢重编程与应激反应通路的一个重要靶点。
SDHC 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 SDHC 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SDHC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SDHC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。