Tailor-made mutations in Arabidopsis using zinc finger nucleases

Methods Mol Biol. 2014:1062:193-209. doi: 10.1007/978-1-62703-580-4_10.

Abstract

Zinc finger nucleases (ZFNs) are proteins engineered to make site-specific double-strand breaks (DSBs) in a DNA sequence of interest. Imprecise repair of the ZFN-induced DSBs by the nonhomologous end-joining (NHEJ) pathway results in a spectrum of mutations, such as nucleotide substitutions, insertions, and deletions. Here we describe a method for targeted mutagenesis in Arabidopsis with ZFNs, which are engineered by context-dependent assembly (CoDA). This ZFN-induced mutagenesis method is an alternative to other currently available gene knockout or knockdown technologies and is useful for reverse genetic studies.

Publication types

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

MeSH terms

  • Arabidopsis / genetics*
  • Base Sequence
  • Cloning, Molecular
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair
  • DNA Mutational Analysis
  • DNA, Plant / genetics
  • DNA, Plant / metabolism
  • Deoxyribonucleases, Type II Site-Specific / biosynthesis
  • Deoxyribonucleases, Type II Site-Specific / genetics*
  • Molecular Sequence Data
  • Mutagenesis*
  • Plants, Genetically Modified / genetics*
  • Transformation, Genetic
  • Zinc Fingers

Substances

  • DNA, Plant
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific