There is a tight molecular link between a Cas protein and its target site on DNA through the use of a guide RNA in every CRISPR reaction, whether it is one that occurs naturally in bacteria or is induced using CRISPR-Cas gene editing technology. It resembles a ski binding on a nanoscale.
Michelle Wang, the James Gilbert White Distinguished Professor of the Physical Sciences and Howard Hughes Medical Institute Investigator at the College of Arts and Sciences, noted that there must be a balance between being securely bound and getting off at the appropriate time. “The capacity to alter affinity is what we actually desire. We can now adjust the potential for gene editing as a result.”
Porter Hall, a biophysics PhD student in the Wang Lab and the paper’s lead author, believes that a Cas protein interaction can’t be too fleeting. Precise gene editing may not be effective and may even have off-target effects if it cannot stably bind the DNA’s target area. However, Hall said, “if the protein remains there indefinitely, the gene editing process cannot be finished.”
Wang and colleagues provide the first mechanistic explanation of how a motor protein (RNA polymerase) removes a bound dCas, a form of Cas designed to detect a DNA sequence without completing a cut, by examining the precise, molecular-level mechanisms involved in Cas binding to DNA.
This understanding clarifies how to adjust Cas removal, advancing potential CRISPR uses.
The article, titled “Polarity of the CRISPR Roadblock to Transcription,” appeared in Nature Structural & Molecular Biology on December 5. James Inman, Robert Fulbright, and Tung Le, who work in the same lab, as well as Guillaume Lambert, an assistant professor of applied and engineering physics at Cornell Engineering, and Joshua Brewer and Seth Darst from the Rockefeller University, are further contributors.
Gaining a thorough mechanistic knowledge of Cas binding stability is essential if CRISPR technology is to reach its full potential, the researchers stated. This study “highlights the role of the R-loop in dCas binding stability and offers useful mechanistic insights for widespread CRISPR technology applications.”
The Wang Lab studies the motion of motor proteins that move along DNA strands to carry out essential biological functions.
According to Wang, the motor protein RNA polymerase pushes against “roadblocks” as it performs its task of expressing genes by copying DNA to RNA. The barrier in this study was endonuclease-deficient Cas (dCas).
The two DNA strands were previously mechanically separated by the researchers in order to determine the location of the attached dCas protein on the DNA using nanophotonic tweezers. This device is known as a DNA unzipping mapper.
Previous studies demonstrated that a motor protein can only remove dCas from one side (a polarity). The reason was that RNA polymerase can only collapse the “R-loop” formed between the guide RNA and the target DNA of a bound dCas from one side, the side distal (or distant) from the PAM (protospacer adjacent motif), a brief DNA sequence 2–6 base pairs long that follows the DNA region targeted for cleavage, according to the Cornell researchers who used the unzipping mapper for the current study.
After describing the method, the researchers also demonstrate how altering the guide RNA may be used to adjust the stability of the dCas R-loop.
In the end, Wang stated, “We expect that fundamental understanding of how Cas proteins function can lead to more effective gene editing and broader uses of the CRISPR technology.”
