Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Biochemistry, Biophysics, and Structural Biology | Cell Biology | Computational Biology | Genetics and Genomics
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, Genetics, 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
J Cell Biol. 2016 Aug 29;214(5):529-37. Epub 2016 Aug 22. Link to article on publisher's site
The Journal of cell biology
Ma H, Tu L, Naseri A, Huisman M, Zhang S, Grünwald D, Pederson T. (2016). CRISPR-Cas9 nuclear dynamics and target recognition in living cells. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1083/jcb.201604115. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1258
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.