In vivo assembly of DNA-fragments in the moss, Physcomitrella patens

Sci Rep. 2016 Apr 29:6:25030. doi: 10.1038/srep25030.

Abstract

Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bryopsida / genetics*
  • DNA, Plant / genetics*
  • Gene Editing
  • Gene Knockout Techniques
  • Homologous Recombination*
  • Promoter Regions, Genetic

Substances

  • DNA, Plant