CRISPR off-target effects explained by DNA coiling

The spatial arrangement of DNA in a cell influences the rate of unintended, off-target effects of CRISPR/Cas9 genome editing.

CRISPR/Cas9 is an approach to genome editing that relies on user-specified sequences of DNA. Off-target editing is always observed to some extent, leading to DNA being altered in unintended locations. This represents a major barrier to applying the technology to treat patients (see BioNews 1073, 1150 and 1268). In a study published in Nature, groups at the University of Sheffield, the MRC Laboratory of Medical Sciences (London) and Imperial College London identified an important factor impacting CRISPR/Cas9 accuracy: how DNA coils on itself in cells.

'It's a tool that is not perfect and can introduce errors and make edits where it shouldn’t make them,' said Professor David Rueda, one of the corresponding authors. 'It’s been estimated to be $0.3 to $0.9 billions per year in industry costs, in profiling off-targets, redesigning guides and delays.'

Efforts to improve CRISPR/Cas9 reliability have previously focused on designing alternative versions of Cas, the enzyme that cuts DNA once bound to a sequence. However, this research has so far mostly been conducted using linear DNA; in cells, DNA is typically coiled to different degrees, giving it unique characteristics. One type of coiling, called negative supercoiling (-SC), had previously been linked to off-target editing.

To study this further, the researchers developed a system to induce the -SC conformation in tiny loops of DNA called minicircles. Crucially, the size of the minicircles made them compatible with microscopy techniques that can show the structure of Cas as it binds to DNA.

'These minicircles are smaller than anything we’ve been able to previously create, pushing the limits of our microscopy technologies,' said Professor Alice Pyne, who contributed to the study. 'These images have enabled us to see how DNA behaves whilst being edited like never before.'

Using cryo-electron microscopy, the researchers were able to show that, when bound to -SC DNA, Cas9 is oriented in a way that makes it more likely to cut DNA. Cas9 is thus more likely to edit incorrectly bound -SC DNA than linear DNA with the same sequence. Attempts to create enhanced Cas proteins using linear DNA have therefore been based on a less-active configuration of the enzyme, rather than the more physiologically relevant one.

'This study definitely paves the way to generate Cas9 variants that are sensitive to topology,' said lead author Dr Quentin Smith. 'High-fidelity variants were designed using linear DNA structures. But in cells, DNA is supercoiled to different degrees, so you might not get that same reduction in off-target activity in the body that you see in the lab.'

Going forward, the approaches developed in this study could help explore how other types of DNA-binding proteins interact with their targets in more realistic conditions.