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.
Neurexin I(NRXN1)是一种突触前细胞黏附分子,通过与 neuroligin、LRRTM 等突触后伙伴结合,组织突触的形成与成熟。通过可变剪接,Neurexin I 使突触识别程序更加多样化,并影响神经递质释放概率、囊泡对接以及钙依赖性胞吐,从而整合进入调控突触组装与可塑性的通路。在大鼠神经系统模型中,NRXN1 的功能常在兴奋/抑制平衡、环路连接性以及活动依赖性重塑等背景下进行研究。文献报道,neurexin 信号的异常与神经发育及神经精神疾病的机制相关,因此 NRXN1 常用于在体内与体外解析突触易损性和网络表型。
neurexin I 双切酶质粒(r)由一对匹配的质粒组成,专为在 rat 细胞系中对 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。