Researchers have uncovered the underlying mechanism that removes Cas proteins from DNA. The study, published in Nature Structural & Molecular Biology, describes the structural features of Cas-DNA binding and the mechanism of Cas removal from DNA. This could lead to more efficient gene editing approaches using CRISPR-Cas technology. Manipulating the binding dynamics could also widen the application of this gene editing tool.
Cas-DNA binding dynamics
CRISPR-Cas technology was developed from a naturally occurring anti-viral defence mechanism found in bacteria. Cas9 (a nuclease enzyme) forms a complex with RNA strands, which guide the enzyme to cut a target sequence in the viral genome. The bond between the Cas protein and the target DNA, via the guide RNA, is a critical interaction. Transient binding is unstable, resulting in inefficient gene-editing and off-target effects. By contrast, binding that is too strong will anchor the protein to the DNA molecule, resulting in incomplete gene-editing. This important interaction has not been studied in-depth.
Researchers at Cornell University and the Rockefeller University investigated the precise mechanism of Cas-DNA binding at a molecular level. They specifically studied how Cas proteins were removed by RNA polymerase (RNAP) enzymes (a motor protein that transcribes DNA to mRNA).
Michelle Wang, Professor of Physical Sciences in the College of Arts and Sciences at Cornell University said, “There’s a balance between being stably bound and coming off at the right time. What we really want is the ability to modulate the affinity. That gives us the possibility of fine-tuning the gene editing potential.”
RNA polymerase removes dCas from one side
CRISPR technology depends on the specific binding of guide RNA molecules to target sequences in the DNA by recognising a downstream PAM (protospacer adjacent motif) sequence (figure 1). This forms an R-loop between the target DNA, where Cas is bound, and the guide RNA. The DNA-bound Cas protein can dissociate spontaneously or be removed by RNAP.
The researchers used dCas to study Cas-DNA interactions. dCas is a modified version of Cas enzymes that binds DNA but does not cut it.
The team described the mechanism of Cas protein removal by RNAP. They found that RNAP can only remove dCas from the PAM-distal side because it can collapse the R-loop. By contrast, the R-loop is inaccessible from the PAM-proximal side, which prevents RNAP-mediated removal.
The researchers showed that modifying the guide RNA, and subsequently the R-loop, allowed them to manipulate dCas removal.
The findings of this study contribute to the mechanistic understanding of Cas-binding stability and highlight the role of R-loop stability in Cas binding dynamics. This understanding could lead to the development of strategies for modulating binding stability. For example, removal of Cas proteins may determine what type of repair pathway is favoured during gene editing.
Professor Wang said, “We hope that the fundamental knowledge of how Cas proteins work can ultimately lead to more efficient gene editing and broader applications of the CRISPR technology.”