Jensen Kristopher Torp, Fløe Lasse, Petersen Trine Skov, Huang Jinrong, Xu Fengping, Bolund Lars, Luo Yonglun, Lin Lin
Department of Biomedicine, Aarhus University, Denmark.
University of Cambridge, UK.
FEBS Lett. 2017 Jul;591(13):1892-1901. doi: 10.1002/1873-3468.12707. Epub 2017 Jun 28.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) systems have emerged as the method of choice for genome editing, but large variations in on-target efficiencies continue to limit their applicability. Here, we investigate the effect of chromatin accessibility on Cas9-mediated gene editing efficiency for 20 gRNAs targeting 10 genomic loci in HEK293T cells using both SpCas9 and the eSpCas9(1.1) variant. Our study indicates that gene editing is more efficient in euchromatin than in heterochromatin, and we validate this finding in HeLa cells and in human fibroblasts. Furthermore, we investigate the gRNA sequence determinants of CRISPR-Cas9 activity using a surrogate reporter system and find that the efficiency of Cas9-mediated gene editing is dependent on guide sequence secondary structure formation. This knowledge can aid in the further improvement of tools for gRNA design.
成簇规律间隔短回文重复序列（CRISPR）相关蛋白9（CRISPR-Cas9）系统已成为基因组编辑的首选方法，但脱靶效率的巨大差异继续限制其适用性。在这里，我们使用SpCas9和eSpCas9(1.1)变体研究了染色质可及性对靶向HEK293T细胞中10个基因组位点的20个向导RNA（gRNA）的Cas9介导的基因编辑效率的影响。我们的研究表明，常染色质中的基因编辑比异染色质中更有效，并且我们在HeLa细胞和人成纤维细胞中验证了这一发现。此外，我们使用替代报告系统研究了CRISPR-Cas9活性的gRNA序列决定因素，发现Cas9介导的基因编辑效率取决于引导序列二级结构的形成。这些知识有助于进一步改进gRNA设计工具。
