Fig. 2.

Model for the strand-dependent roadblocking by the dCas9 and dCas12a complexes. As RNAP moves forward towards the dCas complex from the PAM-distal end, it weakens the numerous small energetic barriers that form between the guide RNA and DNA. With each step, the dCas-PAM interaction becomes slightly destabilized. As a result, by the time RNAP reaches the PAM end, the final barrier is weak and easily overcome, enabling RNAP to dislodge the dCas complex. Conversely, the dCas-PAM interaction at the PAM-proximal end is fully stabilized by the R-loop, creating a large energetic barrier that RNAP must overcome in a single step