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.
PRDX2 编码过氧化还原蛋白2(peroxiredoxin-2,PRX II),这是一种硫醇依赖性的过氧化物酶,利用硫氧还蛋白系统还原过氧化氢和有机氢过氧化物,以维持细胞氧化还原稳态。PRX II 通过塑造局部 H2O2 梯度来调控氧化还原敏感信号,从而影响 MAPK 和 NF-κB 通路的动态、蛋白质 S-谷胱甘肽化以及氧化应激反应。在人体细胞中,PRX II 通过限制 ROS 驱动的大分子损伤,并调控细胞凋亡与炎症信号,支持红细胞完整性及更广泛的细胞保护程序。PRDX2 的表达或活性改变与多种氧化应激和氧化还原信号失衡情境相关,这些情境见于癌症生物学、神经炎症以及心脏代谢性疾病模型,因此 PRDX2 常被作为氧化还原通路研究中的关键机制节点。
PRX II 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 PRDX2 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PRDX2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PRDX2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。