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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