Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779

ACS Synth Biol. 2018 Apr 20;7(4):962-968. doi: 10.1021/acssynbio.7b00362. Epub 2018 Mar 14.

Abstract

Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free nontransgenic engineered lines. To test this system, we disrupted the nitrate reductase gene ( NR) and subsequently removed the CRISPR episome to generate nontransgenic marker-free nitrate reductase knockout lines (NR-KO).

Keywords: CRISPR/Cas9; Nannochloropsis; algal genetic engineering; episome; gene targeting; marker-free.

Publication types

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

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • DNA, Circular
  • Gene Knockout Techniques
  • Genetic Markers
  • Genetic Vectors
  • Microalgae / genetics*
  • Mutation
  • Nitrate Reductase / genetics
  • Organisms, Genetically Modified
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Saccharomyces cerevisiae / genetics
  • Stramenopiles / genetics*

Substances

  • DNA, Circular
  • Genetic Markers
  • Nitrate Reductase