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Scientific Frontline: Extended "At a Glance" Summary: DNA Topology and CRISPR-Cas9 Off-Target Activity
The Core Concept: The natural supercoiling and physical twisting of DNA inside cells increase its susceptibility to unintentional, off-target cutting by the CRISPR-Cas9 gene-editing system.
Key Distinction/Mechanism: While many high-fidelity CRISPR variants are designed and tested using relaxed, linear DNA in a laboratory setting, cellular DNA is naturally supercoiled. This torsional stress forces the DNA to twist and buckle, which reduces the energy barrier required to unzip the double helix. Consequently, it becomes much easier for the Cas9 enzyme to bind and cut incorrect genetic sequences.
Major Frameworks/Components:
- CRISPR-Cas9 System: A highly programmable precision endonuclease tool used to target and edit specific DNA sequences.
- DNA Topology and Supercoiling: The three-dimensional physical strain and torsional stress exerted on genetic material within a living cell.
- DNA Minicircles: Engineered, microscopic loops of DNA utilized in the study to precisely mimic cellular torsional stress while remaining small enough for observation.
- Advanced Imaging: The combined use of cryo-electron microscopy and high-resolution atomic force microscopy to visualize protein-DNA interactions at near-atomic resolution.
Branch of Science: Molecular Biology, Cellular Biophysics, Genetics, and Bioengineering.
Future Application: The findings lay the groundwork for engineering new, topology-sensitive Cas9 variants. These updated tools will be designed to maintain high fidelity in the naturally supercoiled environment of the human body, paving the way for safer personalized cancer treatments and reliable cures for genetic disorders like sickle cell anemia.
Why It Matters: Misplaced DNA edits severely compromise the safety and efficacy of emerging gene therapies, currently costing billions of dollars in delayed or failed drug development. Understanding the mechanical interaction between CRISPR and DNA strain is a critical milestone in eradicating these off-target errors and advancing viable, safe medical treatments.
A mystery surrounding why powerful gene-editing technology that has driven major advances in medicine over the past decade can sometimes make mistakes, has been solved with the help of scientists from the University of Sheffield.
CRISPR-Cas9, a precision DNA-editing tool, has transformed biology by giving scientists a programmable way to cut and edit DNA. Its ever-growing impact includes groundbreaking therapies for genetic diseases such as sickle cell anemia and an increasing role in personalized cancer treatment and rapid diagnostics. However, the system is not perfect as it can sometimes cut DNA sequences that were not the intended targets. These misplaced DNA edits can compromise safety and efficacy, costing billions each year during the search for new therapies.
Now in a study published in the journal Nature, scientists from the Henry Royce Institute at the University of Sheffield have helped to uncover how the physical twisting of DNA plays an important role in these mistakes. The discovery could lead to new gene-editing technologies that are more accurate and help advance new gene therapies.
Sylvia Whittle, a PhD student in the University of Sheffield’s School of Chemical, Materials and Biological Engineering, said: “By visualizing and quantifying changes in the DNA helical structure, we were able to gain fresh insight into how effective different Cas9 interactions can be. This study lays the foundation for generating Cas9 variants that are less error prone. Achieving that step would have a huge impact on healthcare development.”
In the study, led by the MRC Laboratory of Medical Sciences (LMS) and Imperial College London, the researchers captured never-before-seen interactions between CRISPR and DNA, providing insights that could help eradicate errors altogether.
First, during their PhD at the LMS, Dr Quentin Smith, designed a new experimental system in which DNA minicircles can be deliberately supercoiled. These tiny loops of DNA mimic the torsional stress that DNA experiences inside cells, are small enough to visualize by cryo-electron microscopy and maintain a supercoiled state. By placing DNA under stress, the researchers hoped it could reveal how this causes CRISPR-Cas9 to errors in the editing process.
Quentin was awarded a Royce Equipment Access Scheme grant to visit Sheffield’s Royce Discovery Centre. There, they collaborated with the team at Sheffield to use their high-resolution atomic force microscopy technique to determine the sweet spot where the DNA circles were small enough to visualize and still maintained a supercoiled state.
The Sheffield researchers were able to visualize these supercoiled DNA minicircles down to their helical structure. Combined with cryo-electron microscopy imaging at Imperial College London, this allowed the researchers to see, at near-atomic resolution, how CRISPR-Cas9 gene-editing proteins interact with supercoiled DNA.
The images revealed differences in how Cas9 behaved depending on the DNA sequence and the topology. They revealed that as DNA supercoils inside a cell, it makes it far more susceptible to unintentional cutting - an error in the editing process. Their hypothesis is that as DNA twists and buckles, it reduces the energy required to unzip, lowering the energy barrier for Cas9 binding and cutting, and thereby facilitating off-target activity.
Dr Quentin Smith, lead author of the study from the MRC Laboratory of Medical Sciences (LMS), said: “This study paves the way to generate Cas9 variants that are sensitive to topology. Most high-fidelity variants were designed using linear DNA structures. But in cells, the DNA is supercoiled to different degrees, so you might not get the same reduction in off-target activity in the body that you see in the lab.”
Professor Alice Pyne, Professor of Biophysics at the University of Sheffield, said: “We were able to image DNA minicircles in solution, observing their helical structure as they buckled under stress in their supercoiled state. These minicircles are smaller than anything we’ve been able to previously create, pushing the limits of our microscopy technologies. The images have enabled us to see how DNA behaves whilst being edited like never before.”
Professor David Rueda, Head of the Single Molecule Imaging group at the MRC Laboratory of Medical Sciences and Chair in Molecular and Cellular Biophysics at Imperial College London, said: “It’s amazing, we all take Cas9 for granted and think we know everything about it. But we still haven’t seen the truly active structure. This work takes us one step closer – and it paves the way for developing new, more accurate variants.”
Funding: This study was funded by the Medical Research Council, the Engineering and Physical Sciences Research Council, and a UKRI Future Leaders Fellowship.
Published in journal: Nature
Title: Structural basis of supercoiling-induced CRISPR–Cas9 off-target activity
Authors: Quentin M. Smith, Sylvia Whittle, Ricardo J. Aramayo, Daniel E. Rollins, Adam S. B. Jalal, Deborah I. Egharevba, Kyle L. Morris, Alice L. B. Pyne, and David S. Rueda
Source/Credit: University of Sheffield
Reference Number: mbio033026_01
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