Real-Time Observation of DNA Cleavage by CRISPR-Cas9 Endonuclease Using Pyrene Molecule as a Sensitive Probe for Detecting SUB-NM Structural Change

Abstract

CRISPR/Cas9 system performs its nuclease activity through large-scale structural rearrangements. However, its unique property of not releasing the cleaved product has forbidden the spectroscopic observation of its cleavage event in real-time. Here, we employ pyrene molecules as a novel excimer based probe to detect a small-scale structural change not detectable by other methods and demonstrate its versatility to applicate in spectroscopic tracking of Cas9 nuclease activity in time. In this experiment, we show that the sensitive response of excimer emission to minute structural change enables discrimination of structural heterogeneity of the target nucleic acids, such as cleaved and uncleaved nucleic acids within the DNA/Cas9/gRNA ternary complex. In addition, through the observation of Cas9 nuclease activity in time, we reveal that DNA binding is hardly affected by PAM-distal mismatches, while intrinsic rate of cleavage by Cas9 is dramatically decreased even with a 1-bp mismatch. Based on these results, we believe that pyrene-based probe can be used as a promising spectroscopic tool for studying biomolecular systems, which is affected by sub-nm structural changes.