New insights into Cas9 off-target activity

The paper “DNA stretching induces Cas9 off-target binding and cleavage” was just published in Nature Structural and Molecular Biology. Congratulations to all the authors!

In this paper, scientists at the MRC London Institute of Medical Sciences and AstraZeneca studied how CRISPR-Cas9 differentiates between on-targets and off-targets. They used the C-trap™ optical tweezers – fluorescence microscope to monitor fluorescently labeled Cas9 complex binding to λ-DNA in real time. Their data showed that, while Cas9 binds on-target sites tightly and specifically on DNA that is loose and relaxed, stretching the DNA by increasing the tension induces reversible off-target binding at multiple sites. They found that these off-target binding events are driven by mechanical distortion of the DNA – such as the formation of DNA bubbles – and are guide-RNA-sequence dependent. These results suggest that DNA destabilization during cellular processes, such as transcription or replication, can express off-targets to Cas9 activity.

The time-lapse movie published by Prof. David Rueda shows Cas9 binding on DNA tethered between two optically trapped beads. Stretching the DNA from 5 pN to 50 pN causes multiple off-target binding events.

The C-Trap™ Optical Tweezers – Fluorescence Microscopy used in this paper is made commercially available by LUMICKS. Are you interested in using the instrument for your own research? Please feel free to contact us for a demo or quote.

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DNA/RNA-Protein Interactions

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