CRISPR insight: How to fine-tune the Cas protein’s grip on DNA

At the heart of every CRISPR reaction, whether naturally occurring in bacteria or harnessed by CRIPSR-Cas gene editing technology, is a strong molecular bond of a Cas protein via a guide RNA to its target site on DNA. It’s like a nanoscale ski binding.

“There’s a balance between stably bound and coming off at the right time,” said Michelle Wang, the James Gilbert White Distinguished Professor of the Physical Sciences and Howard Hughes Medical Institute Investigator in the College of Arts and Sciences. “What we really want is the ability to modulate the affinity. That gives us the possibility of fine-tuning the gene editing potential.”

A Cas protein binding can’t be too transient, according to Porter Hall, a biophysics doctoral candidate in the Wang Lab and the lead author of the publication. If it can’t stably bind the target region of the DNA, precise gene editing may not be efficient, potentially leading to off-target effects. “But if the protein stays there forever, then the gene editing process cannot be completed,” Hall said.

Examining the precise, molecular-level mechanisms involved in Cas binding to DNA, Wang and colleagues give the first mechanistic explanation of how a motor protein (RNA polymerase) removes a bound dCas, a version of Cas engineered to recognize a DNA sequence without performing a cut.

This insight reveals how to tune Cas removal, contributing to future CRISPR applications.

“Polarity of the CRISPR Roadblock to Transcription” published Dec. 5 in Nature Structural & Molecular Biology. Other contributors are lab members James Inman, Robert Fulbright and Tung Le, along with collaborators Guillaume Lambert, assistant professor in applied and engineering physics, Cornell Engineering, and Joshua Brewer and Seth Darst from the Rockefeller University.

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