Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9.
TLDR
In this article, the photoreceptor genes COP1/2, COP3 (encoding channelrhodopsin 1 [ChR1]), COP4, COP5, PHOT, UVR8, VGCC, MAT3, and aCRY were used for gene editing in Chlamydomonas reinhardtii.Abstract:
The fast-growing biflagellated single-celled chlorophyte Chlamydomonas reinhardtii is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from Staphylococcus aureus and Streptococcus pyogenes, and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP1/2, COP3 (encoding channelrhodopsin 1 [ChR1]), COP4 (encoding ChR2), COP5, PHOT, UVR8, VGCC, MAT3, and aCRY and created the chr1 chr2 and uvr8 phot double mutants. Characterization of the chr1, chr2, and mat3 mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae.read more
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A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis
Xiaobo Li,Xiaobo Li,Xiaobo Li,Weronika Patena,Weronika Patena,Friedrich Fauser,Friedrich Fauser,Robert E. Jinkerson,Robert E. Jinkerson,Shai Saroussi,Moritz T. Meyer,Nina Ivanova,Jacob M. Robertson,Jacob M. Robertson,Rebecca Yue,Ru Zhang,Ru Zhang,Josep Vilarrasa-Blasi,Tyler M. Wittkopp,Tyler M. Wittkopp,Tyler M. Wittkopp,Silvia Ramundo,Sean R. Blum,Audrey Goh,Matthew Laudon,Tharan Srikumar,Paul A. Lefebvre,Arthur R. Grossman,Martin C. Jonikas +28 more
TL;DR: Generation of a library of 62,389 mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii enables screening for genes required for photosynthesis and the identification of 303 candidate genes.
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Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii
Pierre Crozet,Francisco Navarro,Felix Willmund,Payam Mehrshahi,Kamil Bakowski,Kyle J. Lauersen,María Esther Pérez-Pérez,Pascaline Auroy,Aleix Gorchs Rovira,Susana Sauret-Gueto,Justus Niemeyer,Benjamin Spaniol,Jasmine Theis,Raphael Trösch,Lisa-Desiree Westrich,Konstantinos Vavitsas,Thomas Baier,Wolfgang Hübner,Félix de Carpentier,Mathieu Cassarini,Antoine Danon,Julien Henri,Christophe H. Marchand,Marcello De Mia,Kevin Sarkissian,David C. Baulcombe,Gilles Peltier,José L. Crespo,Olaf Kruse,Poul Erik Jensen,Michael Schroda,Alison G. Smith,Stéphane D. Lemaire +32 more
TL;DR: A Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii is developed, based on Golden Gate cloning with standard syntax, and contains 119 openly distributed genetic parts to allow maximum modularity.
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Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy.
Michele Fabris,Michele Fabris,Raffaela M. Abbriano,Mathieu Pernice,Donna L. Sutherland,Audrey S. Commault,Christopher Hall,Leen Labeeuw,Janice I. McCauley,Unnikrishnan Kuzhiuparambil,Parijat Ray,Tim Kahlke,Peter J. Ralph +12 more
TL;DR: How emerging technologies such as synthetic biology, high-throughput phenomics, and the application of internet of things (IoT) automation to algal manufacturing technology can advance the understanding of algal biology and drive the establishment of an algal-based bioeconomy is discussed.
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Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA
TL;DR: It is reported that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as ∼10% efficiency in C. reinhardtii.
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A Series of Fortunate Events: Introducing Chlamydomonas as a Reference Organism
TL;DR: A historical retrospective of the rise of C. reinhardtii is presented to illuminate its past and present and resources for current and future scientists who may wish to expand their studies to the realm of microalgae are presented.
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