天然的CRISPR-Cas9系统由三部分组成:SpCas9(简称Cas9)、crRNA、tracrRNA。tracrRNA(在CRISPR-Cas9编辑技术中被优化并命名为gRNA scaffold),它负责与Cas9结合,与重复序列具有同源性。crRNA为引导序列,约20个碱基,具有特异性。其中,crRNA和tracrRNA通过局部碱基配对组合并与Cas9结合后,引导Cas9识别切割目标DNA序列 (图1)。...
U6启动子,属于真核生物RNA聚合酶III,无物种特异性。 3.gRNA scaffold gRNA scaffold sequence 是gRNA内负责Cas结合的序列,不包括用于引导Cas9靶向DNA的20bp间隔/靶向序列。 4.CaMV enhancer(CaMV 增强子) 增强子是DNA上一段能与蛋白质结合的区域。与蛋白质结合后,基因的转录作用会增强。 5.Kozak sequence Kozak ...
By introducing an A/G mixed capture sequence into the gRNA scaffold, we demonstrate that gRNA transcripts could be directly reverse transcribed by poly (dT) primer together with the endogenous mRNA, followed by high-content molecular phenotyping in scRNA-seq (Direct-seq). With this method, the ...
This study highlights the importance of shortening the 4T sequences in the gRNA scaffold to optimize gRNA transcript expression for enhanced CRISPR-Cas9 gene editing efficiency. This optimization is particularly important for therapeutic applications, where the quantity of vector is often limited, ...
Guide RNA scaffold variants enabled easy cloning of large gRNA cluster for multiplexed gene editingdoi:10.1111/pbi.14198Wang, YaqiLi, XiaofeiLiu, MingleiZhou, YingjiaLi, FengPlant Biotechnology Journal