title: Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9.
abstract: 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.
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Qintao
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China.|State Key Laboratory of Marine Resource Utilization in South China Sea, College of Oceanology, Hainan University, 570228, Haikou, Hainan, China.
authors.forename: Yandu
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xin
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wei
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Shi
authors.initials: S
authors.lastname: Huang
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D058086:Microalgae / Q000235:genetics*; D050901:Nitrate Reductase / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction
chemical_list: D050901:Nitrate Reductase
keywords: Nannochloropsis spp.; clustered regularly interspaced short palindromic repeat-Cas system; genome editing; nitrate reductase; oleaginous microalgae; technical advance