Rationally engineered Cas9 nucleases with improved specificity

Details

• Alternative Title:
  Science
• Personal Author:
  Slaymaker, Ian M. ; Gao, Linyi ; Zetsche, Bernd ; Scott, David A. ; Yan, Winston X. ; Zhang, Feng
• Description:
  The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.
• Subjects:
  Article Bacterial Proteins DNA Cleavage Endonucleases Humans Mutagenesis Point Mutation Protein Conformation Protein Engineering RNA, Guide Streptococcus Pyogenes

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• Source:
  Science. 351(6268):84-88.
• Pubmed ID:
  26628643
• Pubmed Central ID:
  PMC4714946
• Document Type:
  Journal Article
• Funding:
  1R01MH110049/MH/NIMH NIH HHS/United States ; 5DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; 5R01DK097768-03/DK/NIDDK NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; T32GM007753/GM/NIGMS NIH HHS/United States
• Volume:
  351
• Issue:
  6268
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:7b72e916acd856eb28b95473eb757aad17319c256c9e3de0de97a90fd092d515
• Download URL:
  https://stacks.cdc.gov/view/cdc/37554/cdc_37554_DS1.pdf
• File Type:
  [PDF - 1.18 MB ]