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.
SCML2(sex comb on midleg-like 2)是一种多梳(Polycomb)基因家族蛋白,可通过与染色质结合并协调转录抑制程序,参与表观遗传层面的基因调控。在人类细胞中,SCML2在男性生殖系中富集,被认为参与建立支持精子发生的染色质状态,包括对减数分裂相关基因表达的调控以及与性染色体相关的染色质动态变化。SCML2通过调节Polycomb相关通路及组蛋白修饰图谱,影响发育相关基因网络与细胞命运/身份的决定。SCML2功能或表达的异常与生殖细胞发育缺陷有关,并且在研究肿瘤相关转录程序中所见的表观遗传调控失衡时,也被纳入探讨范围。
SCML2 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 SCML2 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SCML2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SCML2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。