首先,作者将负责切割DNA的saCas9与负责靶向lgH位点的sgRNA编码到同一个AAV中;负责提供同源修复的编码bNAb的Doner模版,被构建到另一个AAV中(Figure 1AB)。这样,剪接供体序列中的3BNC117 bNAb基因片段,在整合到位点和随后的转录和剪接后,允许其与恒定的IgH外显子融合,可以破坏内源性IgH位点和bNAb作为膜性BCR的初...
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The compact size of Staphylococcus aureus (SaCas9) gene (3.2 kb) allows it to be packaged into a single AAV vector along with sgRNA expression cassettes, enabling in vivo gene editing applications. For example, a single AAV vector containing SaCas9 and sgRNAs targeting PCSK9 in the mouse liv...
更重要的是,结膜下注射双AAV病毒载体递送的SpCas9/sgRNA-VEGFA系统,可以阻断角膜缝线诱导的VEGFA、CD31和α-平滑肌肌动蛋白的表达,以及小鼠角膜新生血管的形成。这项研究首次为通过基因编辑方法治疗角膜新生血管奠定了坚实基础。该研究进一...
We have further expanded the idea of using AAV to deliver CRISPR/sgRNA in mice, and in turn assessing AAV transduction based on the outcome of CRISPR/sgRNA-mediated genome editing in vivo. We sought to develop an approach to assess AAV transduction in multiple cell types in the CNS by using...
S4c), indicating that the amount of the expressed sgRNA contributes to the genome editing efficiency. Because of the superior performance of AAV9-3sgPKCα-1, all subsequent experiments used this construct (schematic presentation in Fig. 2a). Transduction efficiency using AAV9 + neuraminidase ...
比如在AAV肝脏研究应用中,Wang等人利用AAV-CRISPR介导的直接法体内筛选技术[8],绘制小鼠肝脏肿瘤抑制子的功能性癌症基因组图谱,他们选择U6启动子,使用AAV9- CRISPR-mTSG sgRNA作为载体,通过尾接脉注射利用AAV在Cre依赖CRISPR–Cas9小鼠的肝脏内介导单向导RNA(sgRNA) library表达,进行诱变。注射后0-4个月后进行取材切...
更重要的是,结膜下注射双AAV病毒载体递送的SpCas9/sgRNA-VEGFA系统,可以阻断角膜缝线诱导的VEGFA、CD31和α-平滑肌肌动蛋白的表达,以及小鼠角膜新生血管的形成。这项研究首次为通过基因编辑方法治疗角膜新生血管奠定了坚实基础。 该研究进...
(sgRNA). In addition to the sgRNA, Cas9 requires the presence of a protospacer adjacent motif (PAM), which is a specific sequence proximal to the sgRNA-complementary target sequence1,2,14,15. Once cleaved, the cell relies on the DNA repair machinery to resolve these DSBs through non-...
These constructs were then transfected into HEK293T cells along with one of two A-rich sgRNAs targeting the GAPDH locus (Supplementary Table S4). After 3 days, genomic DNA was harvested and the editing rates of each of the As within the protospacer were evaluated for each construct (...