Conventional RNA-seq and 3'end-seq data from HEK293T. Conventional RNA-seq and 3'end-seq data from HEK293T

To inverstigate off-target effects of CRISPR-iPAS, we transfected HEK293TAPA reporter cells with dPguCas13b and gUSE (showed most effective APA-interference) or non-targeting gRNA (gNT) respectively. 3'end-seq and RNA-seq were performed to investigate if the global change on the endogenous PAS usage and gene expression caused by CRISPR-iPAS. Comparing with gNT, dPguCas13b with gUSE showed high specificity. Overall design: 3'end-seq and RNA-seq were performed on HEK293TAPA cells co-transfected with dPguCas13b and gUSE or gNT respecitively.

为探究CRISPR-iPAS的脱靶效应，本研究分别将dPguCas13b与gUSE（其介导的可变多聚腺苷酸化（APA）干扰效果最为显著），或非靶向向导RNA（gNT）转染至HEK293TAPA报告细胞系中。随后通过3'端测序（3'end-seq）与RNA测序（RNA-seq），探究CRISPR-iPAS是否会对内源多聚腺苷酸位点（PAS）的使用模式及基因表达产生全局性改变。与非靶向向导RNA组（gNT）相比，共转染dPguCas13b与gUSE的样本展现出较高的特异性。整体实验设计：分别对共转染dPguCas13b与gUSE，或共转染dPguCas13b与非靶向向导RNA（gNT）的HEK293TAPA报告细胞系进行3'端测序（3'end-seq）与RNA测序（RNA-seq）。