MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits

Nat Commun. 2020 Nov 27;11(1):6082. doi: 10.1038/s41467-020-19842-2.

Abstract

Gene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9's homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a "plug and play" module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • CRISPR-Associated Protein 9 / chemistry
  • CRISPR-Associated Protein 9 / metabolism*
  • Cell Line
  • Gene Editing*
  • Gene Knock-In Techniques*
  • Green Fluorescent Proteins / metabolism
  • Humans
  • INDEL Mutation / genetics*
  • Rad51 Recombinase / metabolism
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • Vascular Endothelial Growth Factor A
  • Green Fluorescent Proteins
  • Rad51 Recombinase
  • CRISPR-Associated Protein 9