CRI SPR-Cas9 nuclear dynamics and target recognition in living cells.

The bacterial CRI SPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRI SPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRI SPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRI SPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time. [ABSTRACT FROM AUTHOR]