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.
APCS 编码血清淀粉样 P 成分(SAP),它属于五聚蛋白(pentraxin)家族的模式识别蛋白,在血浆中循环,并以钙依赖方式与多种配体结合,包括 DNA、染色质以及微生物多糖。SAP 通过参与补体相关过程以及 Fc 受体介导的清除通路,促进先天免疫监视与调理作用(opsonization),并且在炎症反应期间可与细胞外基质成分发生关联。由于 SAP 能够高亲和力结合淀粉样纤维以及其他错误折叠蛋白聚集体,它被广泛用作研究淀粉样生物学、蛋白沉积以及组织中细胞外聚集体持续存在的分子“抓手”。在系统性炎症和淀粉样相关病理背景下,人们也研究了 SAP 相互作用的改变以及与 APCS 相关的调控变化,从而为免疫复合物处理机制与聚集体稳定化提供机制层面的研究依据。
SAP 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 APCS 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APCS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APCS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。