AbstractNon-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 RNA polymerase II transcriptional pausing followed by transcription termination and potential alternative polyadenylation. By contrast, alternative splicing is unaffected, likely requiring more sustained alteration to 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 the 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 elongation by RNA polymerase II and coupled pre-mRNA processing.