Genome engineering using the CRISPR-Cas9 system

Details

• Alternative Title:
  Nat Protoc
• Personal Author:
  Ran, F. Ann ; Hsu, Patrick D. ; Wright, Jason ; Agarwala, Vineeta ; Scott, David A. ; Zhang, Feng
• Description:
  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.
• Subjects:
  Article Base 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

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• Source:
  Nat Protoc. 2013; 8(11):2281-2308.
• Pubmed ID:
  24157548
• Pubmed Central ID:
  PMC3969860
• Document Type:
  Journal Article
• Funding:
  1DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; 1R01-DK097768/DK/NIDDK NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; F32 DK096822/DK/NIDDK NIH HHS/United States ; R01 DK097768/DK/NIDDK NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; T32GM007753/GM/NIGMS NIH HHS/United States ; T32GM008313/GM/NIGMS NIH HHS/United States
• Volume:
  8
• Issue:
  11
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:fce185f2f74d65793c9d4617959041f0e00e511b767e00fcd4a866f2286e4a62
• Download URL:
  https://stacks.cdc.gov/view/cdc/29900/cdc_29900_DS1.pdf
• File Type:
  [PDF - 1.94 MB ]