How to adjust the Cas protein’s grip on DNA – ScienceDaily

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

“There’s a balance between hard correlation and being out in time,” said Michelle Wang, James Gilbert White Distinguished Professor of Physical Sciences and Howard Hughes Medical Institute Investigator in the College of Arts and Sciences. “What we really want is the ability to modulate affinity. This gives us the possibility to tune gene-editing potential.”

Cas protein binding cannot be transient, according to Porter Hall, a doctoral candidate in biophysics in Wang’s lab and lead author of the publication. If it cannot stably bind the target region of DNA, precise gene editing may not be effective, which can lead to off-target effects. “But if the protein stays there forever, the gene-editing process is not complete,” Hall said.

In examining the precise mechanisms at the molecular level involved in the binding of Cas to DNA, Wang and colleagues provide the first mechanistic explanation for how a motor protein (RNA polymerase) is removed from bound dCas, a version of Cas that is engineered to recognize a DNA sequence without making a cut.

This insight reveals how Cas removal may be tuned, and contributes to future CRISPR applications.

“CRISPR Roadblock to Transcription Polarity” Posted Dec 5 in Structural nature and molecular biology. Other contributors are lab members James Inman, Robert Fulbright, and Tong Lo, along with collaborators Guillaume Lambert, assistant professor of applied and engineering physics, Cornell Engineering, and Joshua Brewer and Seth Darst of The Rockefeller University.

“To fully realize the potential of CRISPR technology, it is essential to gain an in-depth mechanistic understanding of Cas binding stability,” the researchers wrote. “This work highlights the importance of the R-loop in dCas binding stability and provides valuable mechanistic insights for the broad applications of CRISPR technology.”

Wang’s lab is looking at how motor proteins move as they travel along strands of DNA, carrying out vital biological processes.

The RNA polymerase that drives it exerts force on “roadblocks” as it performs its job of gene expression, transcribing DNA into RNA, Wang said. In this study, the barrier was the endonuclease-deficient Cas (dCas).

Previously, using nanophotonic tweezers, researchers mechanically separated DNA strands to locate the dCas-binding protein on the DNA. They call this the DNA unwinding scheme.

Previous research demonstrated that removal of dCas by a motor protein is only unilateral (polarity). Using the decompression scheme of the current study, the Cornell researchers discovered why: Because RNA polymerase can break the loop formed between guide RNA and target DNA (called the “R-loop”) of dCas bound on only one side, the distal (or distal) side of the PAM (Primary Adjacent Form), a short DNA sequence 2–6 base pairs long, follows the target DNA region for cleavage.

Once the researchers describe how the mechanism works, they also show how to adjust dCas R loop stability by modifying guide RNA.

“We hope that basic knowledge of how Cas proteins work will eventually lead to more efficient gene editing and broader applications of CRISPR technology,” Wang said.

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Materials Introduction of Cornell University. Original by Kate Blackwood, courtesy of the Cornell Chronicle. Note: Content can be modified by style and length.

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