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

Overview
TitleNontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779
AuthorsPoliner E, Takeuchi T, Du ZY, Benning C, Farré EM
TypeJournal Article
Journal NameACS Synthetic Biology
Volume7
Issue4
Year2018
Page(s)962-968
CitationPoliner E, Takeuchi T, Du ZY, Benning C, Farré EM. Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779. ACS Synthetic Biology. 2018 04 20; 7(4):962-968.

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).

Properties
Additional details for this publication include:
Property NameValue
Publication TypeJournal Article
Publication ModelPrint-Electronic
Publication Date2018 04 20
Language Abbreng
LanguageEnglish
Journal CountryUnited States
Journal AbbreviationACS Synth Biol
ISSN2161-5063
Elocation10.1021/acssynbio.7b00362
eISSN2161-5063
DOI10.1021/acssynbio.7b00362
Publication TypeResearch Support, N.I.H., Extramural
Publication TypeResearch Support, U.S. Gov't, Non-P.H.S.
Cross References
This publication is also available in the following databases:
DatabaseAccession
PMID: PubMedPMID:29518315