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Nat Protoc DOI:10.1038/nprot.2013.143

Genome engineering using the CRISPR-Cas9 system.

Publication TypeJournal Article
Year of Publication2013
AuthorsF Ran, A, Hsu, PD, Wright, J, Agarwala, V, Scott, DA, Zhang, F
JournalNat Protoc
Date Published2013 Nov
KeywordsBase Sequence, Cell Culture Techniques, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, Deoxyribonucleases, DNA End-Joining Repair, DNA Mutational Analysis, DNA Repair, Genetic Engineering, Genome, Genotyping Techniques, HEK293 Cells, Humans, Molecular Sequence Data, Mutagenesis, Transfection

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.


Alternate JournalNat Protoc
PubMed ID24157548
PubMed Central IDPMC3969860
Grant List1R01-DK097768 / DK / NIDDK NIH HHS / United States
T32 GM007753 / GM / NIGMS NIH HHS / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
F32 DK096822 / DK / NIDDK NIH HHS / United States
T32GM008313 / GM / NIGMS NIH HHS / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
T32 GM008313 / GM / NIGMS NIH HHS / United States
T32GM007753 / GM / NIGMS NIH HHS / United States
1DP1-MH100706 / DP / NCCDPHP CDC HHS / United States