Details

Autor(en) / Beteiligte

• Wang, Qintao
• Lu, Yandu
• Xin, Yi
• Wei, Li
• Huang, Shi
• Xu, Jian

Titel

Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9

Ist Teil von

• The Plant journal : for cell and molecular biology, 2016-12, Vol.88 (6), p.1071-1081

Ort / Verlag

England: Blackwell Publishing Ltd

Erscheinungsjahr

2016

Quelle

MEDLINE

Beschreibungen/Notizen

• Summary Microalgae are promising feedstock for biofuels yet mechanistic probing of their cellular network and industrial strain development have been hindered by lack of genome‐editing tools. Nannochloropsis spp. are emerging model microalgae for scalable oil production and carbon sequestration. Here we established a CRISPR/Cas9‐based precise genome‐editing approach for the industrial oleaginous microalga Nannochloropsis oceanica, using nitrate reductase (NR; g7988) as example. A new screening procedure that compares between restriction enzyme‐digested nested PCR (nPCR) products derived from enzyme‐digested and not‐digested genomic DNA of transformant pools was developed to quickly, yet reliably, detect genome‐engineered mutants. Deep sequencing of nPCR products directly amplified from pooled genomic DNA revealed over an 1% proportion of 5‐bp deletion mutants and a lower frequency of 12‐bp deletion mutants, with both types of editing precisely located at the targeted site. The isolated mutants, in which precise deletion of five bases caused a frameshift in NR translation, grow normally under NH4Cl but fail to grow under NaNO3, and thus represent a valuable chassis strain for transgenic‐strain development. This demonstration of CRISPR/Cas9‐based genome editing in industrial microalgae opens many doors for microalgae‐based biotechnological applications. Significance Statement Microalgae are promising feedstocks for biofuels, but additional genomic tools are needed to fully dissect the cellular networks underlying production of oil and biomass. Here, using nitrate reductase as an example, we demonstrate CRISPR/Cas9‐based genome editing for a model industrial microalga.