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

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 NH