Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
The bacterial CRISPR-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 (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR 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 time.
- DNA Biology,
- RNA Biology
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© 2016 Ma et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
DOI of Published Version
Creative Commons License
Creative Commons Attribution-Noncommercial-Share Alike 3.0
Hanhui Ma, Li-Chun Tu, Ardalan Naseri, Maximiliaan Huisman, et al.. "CRISPR-Cas9 nuclear dynamics and target recognition in living cells" Vol. 214 Iss. 5 (2016) ISSN: 0021-9525 (Linking)
Available at: http://works.bepress.com/david_grunwald/26/