Distorted DNA may increase risk of changes with CRISPR-Cas9

Distortions to DNA, which occur routinely during gene expression and other cellular processes, could lead to off-target changes to the genome when using CRISPR-Cas9, a new study suggests. The Medical Research Council scientists behind the research say that their findings may help to pave the way to improve the accuracy of gene editing for clinical applications.

During the expression of genes, DNA is stretched and distorted out of its usual shape. While this is needed for proper function of the cell’s own machinery, it may pose a challenge for CRISPR-Cas9 gene editing by increasing the risk of off-target edits, potentially introducing harmful changes. The findings, from scientists at the MRC London Institute of Medical Sciences and AstraZeneca, are published today in the journal Nature Structural and Molecular Biology.

CRISPR-Cas9, a gene editing tool that allows researchers to find and edit strands of DNA, has gained worldwide recognition for its multitude of purposes as scientists use the technology across a range of sectors, including medicine, drug discovery and agriculture.

In the study, the accuracy and precision of CRISPR-Cas9 was investigated using a novel approach: scientists used optical tweezers – a tool that uses laser beams to manipulate DNA – to mimic the contortions that DNA naturally goes through as it is read by the cell’s machinery. They then used CRISPR-Cas9 to edit the gene and monitored its accuracy using fluorescence microscopy.

Results showed that CRISPR is accurate when DNA is loose and relaxed. But when distortions occurred – in this case from being highly stretched – accuracy decreased, and off-target edits were observed. Understanding this effect will aid the design of CRISPR systems with increased accuracy, alongside methods to assess this risk.