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介导的多聚腺苷酸位点干扰（CRISPR-iPAS）的脱靶效应（off-target effects），我们分别将失活型PguCas13b（dPguCas13b）、展现出最优多聚腺苷酸化（APA）干扰效果的gUSE，以及非靶向向导RNA（non-targeting gRNA, gNT）转染至HEK293TAPA报告细胞系（HEK293TAPA reporter cells）中。随后通过3'端测序（3'end-seq）与RNA测序（RNA-seq），以探究CRISPR-iPAS是否会对内源多聚腺苷酸位点（polyadenylation site, PAS）使用及基因表达产生全局改变。与非靶向向导RNA对照组相比，搭载gUSE的失活型PguCas13b展现出较高的特异性。整体实验设计：分别对共转染失活型PguCas13b与gUSE或gNT的HEK293TAPA细胞开展3'端测序与RNA测序。