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.
人类 PRDX4 基因编码过氧化物还原蛋白 IV(PRX IV)。PRX IV 是一种位于内质网及分泌途径的、依赖巯基的过氧化物酶,可将过氧化氢和脂质过氧化物还原,从而维持细胞氧化还原稳态。通过将过氧化物解毒与二硫键形成相偶联,PRX IV 支持氧化型蛋白折叠、内质网蛋白质稳态(ER proteostasis),并在氧化应激下减弱未折叠蛋白反应(UPR)信号。PRDX4 的活性与多条受氧化还原调控的通路相交,参与分泌、炎症与细胞生存的调控;其表达水平或氧化状态的改变,已与多种氧化应激表型相关,这些表型见于癌症生物学、代谢功能紊乱以及心血管与炎症相关情境。作为一种分泌型/内质网氧化还原酶,PRX IV 常被用于研究其对细胞外及腔内活性氧(ROS)缓冲的影响,以及由此引发的应激适应性转录程序的下游调控。
PRX IV 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 PRDX4 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PRDX4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PRDX4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。