High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets

Details

• Alternative Title:
  Nature
• Personal Author:
  Kleinstiver, Benjamin P. ; Pattanayak, Vikram ; Prew, Michelle S. ; Tsai, Shengdar Q. ; Nguyen, Nhu ; Zheng, Zongli ; Joung, J. Keith
• Description:
  CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
• Subjects:
  Base Sequence Clustered Regularly Interspaced Short Palindromic Repeats CRISPR-Associated Proteins CRISPR-Cas Systems DNA DNA-Binding Proteins Endonucleases Genetic Engineering Genome, Human Humans Mutation Protein Binding Reproducibility Of Results RNA Sequence Analysis, DNA Streptococcus Pyogenes Substrate Specificity

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• Source:
  Nature. 529(7587):490-495.
• Pubmed ID:
  26735016
• Pubmed Central ID:
  PMC4851738
• Document Type:
  Text
• Funding:
  DP1 GM105378/DP/NCCDPHP CDC HHS/United States ; DP1 GM105378/GM/NIGMS NIH HHS/United States ; R01 GM088040/GM/NIGMS NIH HHS/United States ; R01 GM107427/GM/NIGMS NIH HHS/United States
• Genre:
  Journal Article
• Volume:
  529
• Issue:
  7587
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha-512:f014c0da4a6db327fd450a7525c3164cb4e9ff4de4dc9fee459e84455e7b836cd2ab16de405432101eda068ecd7690f8271ae1cc14cf6c688ca61ebc61fa92df
• Download URL:
  https://stacks.cdc.gov/view/cdc/39163/cdc_39163_DS1.pdf
• File Type:
  [PDF - 4.90 MB ]