Genome engineering using the CRISPR-Cas9 system
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Zhang_Genome engineering.pdf
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Author(s)
Ran, F. Ann
Hsu, Patrick
Wright, Jason
Agarwala, Vineeta
Scott, David Arthur
Zhang, Feng
Date Issued
November 2013
Journal
Nature Protocols
Publisher
Nature Publishing Group
Citation
Ran, F Ann, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott, and Feng Zhang. “Genome Engineering Using the CRISPR-Cas9 System.” Nature Protocols 8, no. 11 (October 24, 2013): 2281–2308.
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Author's final manuscript
Abstract
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks.
Description
available in PMC 2014 March 30
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
McGovern Institute for Brain Research at MIT
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1038/nprot.2013.143
