GENOME EDITING IN HUMAN CELLS USING CRISPR/CAS NUCLEASES

Details

• Alternative Title:
  Curr Protoc Mol Biol
• Personal Author:
  Wyvekens, Nicolas ; Tsai, Shengdar ; Joung, J. Keith
• Description:
  The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases.
• Subjects:
  Article Cas9 CRISPR FokI-dCas9 Genome Editing Human Cells RNA-guided FokI Nucleases Tru-gRNA
• Source:
  Curr Protoc Mol Biol. 112:31.3.1-31.318.
• Pubmed ID:
  26423589
• Pubmed Central ID:
  PMC4852847
• Document Type:
  Journal Article
• Funding:
  DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; DP1 GM105378/GM/NIGMS NIH HHS/United States ; F32 GM105189/GM/NIGMS NIH HHS/United States ; P50 HG005550/HG/NHGRI NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States
• Volume:
  112
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:043681f6ae0ef452bbdfa42939b11028c10de689497beec12e77ebaebda4fe55
• Download URL:
  https://stacks.cdc.gov/view/cdc/39220/cdc_39220_DS1.pdf
• File Type:
  [PDF - 1.05 MB ]