CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.

基于 CRISPR 的系统从根本上改变了我们研究和操作干细胞的能力。我们探索了使用来自化脓性链球菌的催化死亡 Cas9 (dCas9) 的可能性作为在干细胞中进行靶向表观遗传编辑的平台，以增强分化过程中初中胚层基因 (EOMES) 的表达。然而，我们观察到 dCas9 蛋白本身在 hiPSC 中发挥潜在的非特异性作用，在随后的定向分化过程中影响细胞的表型和基因表达模式。我们表明，当细胞在不积极维持多能网络的培养基中培养时，这种效应是特定的，并且不含 sgRNA 的 apo-dCas9 蛋白本身会影响内源性基因表达。转录组学分析显示，大量参与发育过程的基因和具有非重叠生物学功能的各种其他基因受到 dCas9 过表达的影响。