![]() The red arrows indicate locations where the unzipping force dipped below the naked DNA baseline. The two dashed lines bracket the expected gRNA-DNA hybrid locations for dCas9 or dCas12a and the expected RNA-DNA hybrid of a TEC. Two conformations were detected when a bound dCas12a protein was unzipped from the PAM-distal side, shown as light blue and red. The DNA was unzipped from either direction (black arrows) relative to the bound protein for each protein. b, Representative unzipping traces (red) of bound dCas9 (top), bound dCas12a (middle) and a paused TEC (bottom), along with naked DNA traces (black). The location of the rise in force is used to map the protein location. Using the optical trap, the bead is moved relative to the surface, progressively unzipping the DNA until the unzipping fork reaches a bound protein, which resists unzipping, leading to a distinct rise in force. An unzipping template is tethered at one end to the surface of a coverslip of a sample chamber and at the other end to a polystyrene bead held in an optical trap. This mechanistic understanding suggests strategies for modulating dCas stability and holds broader implications for Cas applications.Ī, DNA unzipping mapper configuration. Through this, we discovered the mechanism for CRISPRi polarity and dCas removal, highlighting the importance of the R-loop stability for a bound Cas. Using single-molecule assays, we mapped the structural features of a dCas complex bound to DNA and investigated how an elongating RNAP interacts with the bound dCas. The CRISPRi system offers an appealing opportunity to examine the polarity and mechanics of dCas removal and, more broadly, Cas-binding stability. Curiously, a bound dCas is not found to be a polar barrier to replication 9, 10, indicating that the polarity is dictated by the dynamics of how motor proteins overcome dCas barriers. Transcription elongation is rather permissive from the PAM-distal side of a bound dCas but is predominantly blocked from the PAM-proximal side 6, 7, 8. Intriguingly, during CRISPR interference (CRISPRi), which uses a dCas to block transcription, the effectiveness of dCas removal depends on the orientation of the bound dCas relative to transcription. However, the mechanism governing Cas removal by motor proteins is not well understood. In vivo, a DNA-bound Cas can not only dissociate from the DNA spontaneously but also be removed by motor proteins carrying out other host processes. This occurs via recognition of a PAM sequence and hybridization of the spacer region of the gRNA with the target DNA to form a gRNA-DNA hybrid (R-loop) 1, 5. A critical facet of CRISPR utility relies on Cas enzyme-binding stability, which is dictated by specific and robust binding of the gRNA to the target DNA sequence. To accomplish this, a Cas protein is complexed with a guide RNA (gRNA) that contains a spacer region complementary to the target DNA sequence. The utilization of CRISPR-associated (Cas) nucleases offers the ability to precisely target DNA sequences and cleave at those sites, enabling great advances in gene editing, targeting and diagnostic technology for both prokaryotic and eukaryotic systems 1, 2, 3, 4. This work highlights the importance of the R-loop in dCas-binding stability and provides valuable mechanistic insights for broad applications of CRISPR technology. This mechanistic understanding allowed us to modulate the dCas R-loop stability by modifying the guide RNAs. This finding is not unique to RNAP and holds for the Mfd translocase. By mapping dCas-DNA interactions at high resolution, we discovered that the collapse of the dCas R-loop allows Escherichia coli RNAP read-through from the PAM-distal side for both Sp–dCas9 and As–dCas12a. Intriguingly, during CRISPR interference, RNA polymerase (RNAP) progression is only fully blocked by a bound endonuclease-deficient Cas (dCas) from the protospacer adjacent motif (PAM)-proximal side. However, a Cas complex bound to DNA may be removed by motor proteins carrying out host processes and the mechanism governing this removal remains unclear. ![]() CRISPR (clustered regularly interspaced short palindromic repeats) utility relies on a stable Cas effector complex binding to its target site.
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