Elongation roadblocks mediated by dCas9 across human genes modulate transcription and nascent RNA processing

Non-cleaving Cas9 (dCas9) is widely employed to manipulate specific gene loci often with scant regard for unintended transcriptional effects. We demonstrate here that dCas9 mediates precise transcriptional pausing followed by transcription termination and potential alternative polyadenylation. In contrast, alternative splicing is unaffected, likely requiring more sustained alteration to RNA polymerase II elongation speed. The effect on transcription is orientation-specific, with pausing only being induced when dCas9-associated guide RNA anneals to the non-template strand. Targeting template strand induces minimal effects on transcription elongation and thus provides a neutral approach to recruit dCas9 linked effector domains to specific gene regions. In essence we evaluate molecular effects of targeting dCas9 to mammalian transcription units. In so doing we also provide new information on transcriptional elongation by RNA polymerase II and coupled pre-mRNA processing. Prior to libraries preparation, HeLa cells were transfected with CTRL or gene-specific CRISPRi constructs targeting TXNRD1 intron (in2), TXNRD1 downstream area (DS2) or a set of alternatively polyadenylated gene (APA).