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.
SCGB1A1 编码子宫球蛋白(uteroglobin,也称 CC10/CCSP),它是分泌型 secretoglobin 家族成员,主要由气道的 Club 细胞(又称 Clara 细胞)以及其他黏膜上皮产生。子宫球蛋白具有抗炎、免疫调节及与抗氧化相关的作用,可影响上皮屏障稳态并调控对环境性损伤的应答;其中部分机制与其对细胞因子信号以及类二十烷酸(eicosanoid)相关过程的调控有关。SCGB1A1 的表达常被用作 Club 细胞分化与气道上皮状态的标志物,因此与黏膜防御、组织修复和先天免疫调控等通路相关联。在炎症性气道疾病、烟雾或污染物暴露应答,以及与呼吸系统疾病模型相关的上皮重塑表型研究中,SCGB1A1 水平改变和 Club 细胞功能障碍是常见的研究重点。
Uteroglobin/SCGB1A1/CC10 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 SCGB1A1 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SCGB1A1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SCGB1A1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。