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.
AXL 基因编码 Axl 蛋白,Axl 属于 TAM 家族受体酪氨酸激酶,可被 GAS6 等配体激活,从而调控细胞存活、增殖、迁移以及凋亡细胞清除(efferocytosis)。Axl 信号可激活 PI3K–AKT、MAPK/ERK、JAK/STAT 和 NF-κB 等通路,并调节整合素及细胞骨架动态,进而影响细胞黏附与运动性。在免疫与基质相关环境中,Axl 促进炎症消退并介导对凋亡细胞的吞噬清除,塑造细胞因子信号与组织重塑。多种肿瘤和炎症性疾病模型中,AXL 表达或活性失调与侵袭性表型及免疫微环境改变相关,因此支持对受体信号及通路串扰机制开展研究。
Axl 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 AXL 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AXL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AXL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。