Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

Commun Biol. 2020 Jan 23;3(1):44. doi: 10.1038/s42003-020-0768-9.

Abstract

Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homology-directed repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYNTHASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry
  • ATP-Binding Cassette Transporters / genetics
  • Base Sequence
  • CRISPR-Associated Protein 9 / chemistry
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism*
  • Endodeoxyribonucleases / chemistry
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Gene Editing*
  • Genes, Plant
  • Genetic Engineering* / methods
  • Genome, Plant
  • Herbicide Resistance / genetics
  • Oryza / drug effects
  • Oryza / genetics*
  • Oryza / metabolism
  • Phenotype
  • Protein Binding
  • Recombinant Fusion Proteins*
  • Recombinational DNA Repair*

Substances

  • ATP-Binding Cassette Transporters
  • Recombinant Fusion Proteins
  • CRISPR-Associated Protein 9
  • Endodeoxyribonucleases