DNA Structure Modulates the Specificity of CRISPR Gene Editing Technology

Scientists have revealed that, under certain conditions, changes to cellular DNA structure can result in unwanted off-target effects that should be considered in applying CRISPR gene editing technology.

 20 October 2023  

By Jianyi Nie 

Gene editing is a process developed by scientists which uses precise molecular tools to make changes to an organism’s DNA. It can target specific DNA sequences and effectively cut out and replace entire sections of a gene. This has made it an invaluable tool in genetic research and holds great potential for therapeutic applications – it could in future be used as a tool to fix and repair any genetic disorder by simply editing disease-causing mutations. Currently, it is one of the most promising applications for treating sickle cell anaemia. 

CRISPR-Cas9 is generally seen as a powerful gene-editing tool. However, the safety and accuracy of the technique have been the subject of scrutiny, particularly due to the potential for use in the clinic. Researchers at LMS and collaborators have demonstrated that the CRISPR-Cas9 technique can be more problematic than previously thought- when the DNA is partially unwound, they have shown that Cas9 can become less accurate.  

The study, led by groups at the Medical Research Council Laboratory of Medical Sciences (MRC LMS), with collaborators from AstraZeneca, University College London, The Francis Crick Institute, and Cardiff University, was published on October 6th, 2023 in Molecular Cell. 

“Scientists have long desired to increase the accuracy of CRISPR to enhance its potential for therapeutic use,” said Professor David S. Rueda, the lead author of the study and leader of the MRC LMS Single Molecule Imaging group. “Our study sheds light on a critical aspect of Cas9’s behavior that has been previously overlooked.” 

When the DNA double helix is underwound or twisted in the opposite direction of its natural spiral, it is called negative DNA supercoiling. In this state, the two DNA strands are more separated or uncoiled than they would be in their relaxed state. Scientists explored the interplay between negative DNA supercoiling and Cas9’s off-target activity in this study. 

The LMS researchers used a novel technique to unwind DNA sequences. They found that unwinding the DNA can lead to over 10,000 potential off-target effects genome-wide. This shows that we may need a more in-depth understanding of factors affecting this gene-editing tool. 

They discovered that negative DNA supercoiling could impact the Cas9’s ability to access and interact with specific DNA sequences, potentially leading to more accidental mutations. Furthermore, the research team demonstrated that Cas9 could cut off-target sites with a high number of mismatches when the DNA was negatively supercoiled. Thereby raising concerns about the accuracy of CRISPR. 

“CRISPR offers a precise and efficient way to modify DNA. However, our study shows that CRISPR accuracy is affected by the environment that it’s used in,” said Dr Matthew D. Newton, the first author of the study and who worked on this project during his PhD at the MRC LMS. “Our study provides important knowledge that may help researchers optimize Cas9-based tools and minimize unintended genetic modifications.” 

Read the study here.