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.
AMELY 编码 Y 染色体上的成釉蛋白(amelogenin)同工型,这是一种分泌型细胞外基质蛋白。它通过调控羟基磷灰石晶体的成核、生长以及基质组织,参与牙釉质的生物矿化与牙齿发育。成釉蛋白在成釉过程中参与牙釉质基质的组装与蛋白水解加工,影响上皮–间充质相互作用以及矿物沉积程序。影响成釉蛋白表达或结构的遗传变异已与牙釉质形成缺陷及釉质发育不全谱系疾病中的相关表型相联系,提示其在颅颌面与牙科发育生物学中具有重要意义。由于 AMELY 为 Y 连锁基因且与 AMELX 关系密切,它也可用于研究性染色体生物学以及在成牙模型中旁系同源基因特异性的调控机制。
Amelogenin 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 AMELY 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AMELY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AMELY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。