@article{0520247724a14d7b87f767a9bcd1d19f,
title = "CRISPR deactivation in mammalian cells using photocleavable guide RNAs",
abstract = "The ability to deactivate CRISPR-Cas systems on demand would improve the safety and applicability of genome editing. Here, we detail a protocol using photocleavable guide RNAs (pcRNAs) to deactivate CRISPR-Cas9 inside cells. We verify that deactivation is both rapid and complete by checking for insertion-deletion (indel) mutations using Sanger sequencing. This protocol will be useful for researchers interested in using pcRNAs to improve genome editing specificity, characterize the timescales of genome editing, and study cellular DNA damage responses. For complete details on the use and execution of this protocol, please refer to Zou et al. (2021).",
keywords = "Biotechnology and bioengineering, CRISPR, Cell Biology, Cell-based Assays, Genomics, Molecular Biology, Molecular/Chemical Probes, Sequencing",
author = "Zou, {Roger S.} and Yang Liu and Taekjip Ha",
note = "Funding Information: We thank Dr. Geraldine Seydoux for access to the Lonza 4D nucleofector. This work was supported by the grants from the NIH (R35 GM 122569 to T.H. T32 GM 73009 and F30 CA 254160 to R.S.Z.), NSF (PHY 1430124 to T.H. 1817447 to B.W.), and Pew Charitable Trust (00030601 to B.W.). T.H. is an investigator of the Howard Hughes Medical Institute. R.S.Z. and T.H. initially conceived the project. R.S.Z. and Y.L. performed experiments and analyzed the data. R.S.Z. wrote the manuscript with contributions from all authors. T.H. supervised the project. The authors and Johns Hopkins University have authored patent application PCT/US20/57255, including the work described herein. Funding Information: We thank Dr. Geraldine Seydoux for access to the Lonza 4D nucleofector. This work was supported by the grants from the NIH ( R35 GM 122569 to T.H., T32 GM 73009 and F30 CA 254160 to R.S.Z.), NSF ( PHY 1430124 to T.H., 1817447 to B.W.), and Pew Charitable Trust ( 00030601 to B.W.). T.H. is an investigator of the Howard Hughes Medical Institute. Publisher Copyright: {\textcopyright} 2021 The Author(s)",
year = "2021",
month = dec,
day = "17",
doi = "10.1016/j.xpro.2021.100909",
language = "English (US)",
volume = "2",
journal = "STAR Protocols",
issn = "2666-1667",
publisher = "Cell Press",
number = "4",
}