Predictable and precise template-free CRISPR editing of pathogenic variants

Nature. 2018 Nov;563(7733):646-651. doi: 10.1038/s41586-018-0686-x. Epub 2018 Nov 7.

Abstract

Following Cas9 cleavage, DNA repair without a donor template is generally considered stochastic, heterogeneous and impractical beyond gene disruption. Here, we show that template-free Cas9 editing is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. We constructed a library of 2,000 Cas9 guide RNAs paired with DNA target sites and trained inDelphi, a machine learning model that predicts genotypes and frequencies of 1- to 60-base-pair deletions and 1-base-pair insertions with high accuracy (r = 0.87) in five human and mouse cell lines. inDelphi predicts that 5-11% of Cas9 guide RNAs targeting the human genome are 'precise-50', yielding a single genotype comprising greater than or equal to 50% of all major editing products. We experimentally confirmed precise-50 insertions and deletions in 195 human disease-relevant alleles, including correction in primary patient-derived fibroblasts of pathogenic alleles to wild-type genotype for Hermansky-Pudlak syndrome and Menkes disease. This study establishes an approach for precise, template-free genome editing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alleles
  • Base Sequence
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics*
  • DNA Repair / genetics
  • Fibroblasts / metabolism
  • Fibroblasts / pathology
  • Gene Editing / methods*
  • Gene Editing / standards*
  • HCT116 Cells
  • HEK293 Cells
  • Hermanski-Pudlak Syndrome / genetics*
  • Hermanski-Pudlak Syndrome / pathology
  • Humans
  • K562 Cells
  • Machine Learning*
  • Menkes Kinky Hair Syndrome / genetics*
  • Menkes Kinky Hair Syndrome / pathology
  • Reproducibility of Results
  • Substrate Specificity
  • Templates, Genetic*

Substances

  • CRISPR-Associated Protein 9