title: DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins.
abstract: Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.
authors.affiliation: Department of Life Science, Hanyang University, Seoul, South Korea.
authors.forename: Kwangryul
authors.initials: K
authors.lastname: Baek
authors.affiliation: Department of Chemistry, Hanyang University, Seoul, South Korea.
authors.forename: Duk Hyoung
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Department of Life Science, Hanyang University, Seoul, South Korea.
authors.forename: Jooyeon
authors.initials: J
authors.lastname: Jeong
authors.affiliation: Department of Chemical and Biological Engineering, Korea University, Seoul, South Korea.
authors.forename: Sang Jun
authors.initials: SJ
authors.lastname: Sim
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA.
authors.forename: Anastasios
authors.initials: A
authors.lastname: Melis
authors.affiliation: Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea.|Department of Chemistry, Seoul National University, Seoul, South Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Department of Life Science, Hanyang University, Seoul, South Korea.
authors.forename: EonSeon
authors.initials: E
authors.lastname: Jin
authors.affiliation: Department of Chemistry, Hanyang University, Seoul, South Korea.
authors.forename: Sangsu
authors.initials: S
authors.lastname: Bae
mesh_terms: D020418:Algal Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D060847:Metabolic Engineering; D010788:Photosynthesis; D012261:Ribonucleoproteins / Q000378:metabolism*; D065146:Zeaxanthins / Q000378:metabolism
chemical_list: D020418:Algal Proteins; D012261:Ribonucleoproteins; D065146:Zeaxanthins