Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

Elife. 2018 May 29;7:e33761. doi: 10.7554/eLife.33761.


The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.

Keywords: CRISPR/Cas9; chromosomes; gene editing; gene expression; homology-directed repair; human; human biology; medicine; mouse.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA / chemistry
  • DNA / metabolism*
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair*
  • DNA Replication
  • Gene Editing / methods*
  • Genetic Loci
  • Guanidines / chemistry
  • HEK293 Cells
  • Humans
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism
  • RNA, Guide, Kinetoplastida / genetics
  • RNA, Guide, Kinetoplastida / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombinational DNA Repair*
  • Staining and Labeling / methods


  • Guanidines
  • Oligodeoxyribonucleotides
  • RNA, Guide
  • Recombinant Fusion Proteins
  • benzylguanidine
  • DNA
  • CRISPR-Associated Protein 9