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PMID: 24157548 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Genome engineering using the CRISPR-Cas9 system.

Nature protocols ·Vol. 8 ·No. 11 ·2013-11-00 ·Pages 2281-2308

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F

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.

MeSH Terms
Base Sequence Cell Culture Techniques Cell Line Clustered Regularly Interspaced Short Palindromic Repeats DNA End-Joining Repair DNA Mutational Analysis DNA Repair Deoxyribonucleases/chemistry,genetics Genetic Engineering/methods Genome Genotyping Techniques HEK293 Cells Humans Molecular Sequence Data Mutagenesis Transfection
Chemicals
Deoxyribonucleases
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Ran F Ann
Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA. | McGovern Institute for Brain Research, Cambridge, Massachusetts, USA. | Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA. | Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA. | Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Hsu Patrick D
Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA. | McGovern Institute for Brain Research, Cambridge, Massachusetts, USA. | Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA. | Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA. | Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Wright Jason
Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA.
Agarwala Vineeta
Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA. | Program in Biophysics, Harvard University, MIT, Cambridge, Massachusetts, USA. | Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA.
Scott David A
Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA. | McGovern Institute for Brain Research, Cambridge, Massachusetts, USA. | Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA. | Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.
Zhang Feng
Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA. | McGovern Institute for Brain Research, Cambridge, Massachusetts, USA. | Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA. | Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.
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Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2013.143
Published
2013-11-00
Epub
2013-00-24
Pages
2281-2308
Language
English
Region
England
NLM ID
101284307
PMCID
PMC3969860
Subset
IM
Grants
NIDDK NIH HHS · 1R01-DK097768 · United States
NIGMS NIH HHS · T32 GM007753 · United States
NIMH NIH HHS · DP1 MH100706 · United States
NIGMS NIH HHS · T32GM007753 · United States
NCCDPHP CDC HHS · 1DP1-MH100706 · United States
NIDDK NIH HHS · F32 DK096822 · United States
NIGMS NIH HHS · T32GM008313 · United States
NIDDK NIH HHS · R01 DK097768 · United States
NIGMS NIH HHS · T32 GM008313 · United States
External Links
PubMed source DOI: 10.1038/nprot.2013.143 PMC Full Text View journal details
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