Precise gene replacement in rice by RNA transcript-templated homologous recombination

Nat Biotechnol. 2019 Apr;37(4):445-450. doi: 10.1038/s41587-019-0065-7. Epub 2019 Mar 18.

Abstract

One of the main obstacles to gene replacement in plants is efficient delivery of a donor repair template (DRT) into the nucleus for homology-directed DNA repair (HDR) of double-stranded DNA breaks. Production of RNA templates in vivo for transcript-templated HDR (TT-HDR) could overcome this problem, but primary transcripts are often processed and transported to the cytosol, rendering them unavailable for HDR. We show that coupling CRISPR-Cpf1 (CRISPR from Prevotella and Francisella 1) to a CRISPR RNA (crRNA) array flanked with ribozymes, along with a DRT flanked with either ribozymes or crRNA targets, produces primary transcripts that self-process to release the crRNAs and DRT inside the nucleus. We replaced the rice acetolactate synthase gene (ALS) with a mutated version using a DNA-free ribonucleoprotein complex that contains the recombinant Cpf1, crRNAs, and DRT transcripts. We also produced stable lines with two desired mutations in the ALS gene using TT-HDR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetolactate Synthase / genetics
  • Base Sequence
  • Biotechnology
  • CRISPR-Cas Systems
  • DNA, Plant / genetics
  • Gene Targeting / methods*
  • Genes, Plant*
  • Homologous Recombination*
  • Mutation
  • Oryza / genetics*
  • Plant Proteins / genetics
  • Plants, Genetically Modified
  • RNA, Plant / genetics
  • Recombinant Proteins / genetics
  • Recombinational DNA Repair
  • Templates, Genetic

Substances

  • DNA, Plant
  • Plant Proteins
  • RNA, Plant
  • Recombinant Proteins
  • Acetolactate Synthase