Structure and expression of the gene encoding the periplasmic arylsulfatase of Chlamydomonas reinhardtii

1989 ◽  
Vol 218 (2) ◽  
pp. 229-239 ◽  
Author(s):  
Eugenio L. de Hostos ◽  
James Schilling ◽  
Arthur R. Grossman
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Gene Encoding

scholarly journals Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

2014 ◽  
Vol 13 (11) ◽  
pp. 1465-1469 ◽  
Author(s):  
Wenzhi Jiang ◽  
Andrew J. Brueggeman ◽  
Kempton M. Horken ◽  
Thomas M. Plucinak ◽  
Donald P. Weeks
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Transient Expression ◽  
Gene Knockout ◽  
Nuclease Activity ◽  
Gene Replacement ◽  
Target Site ◽  
Gene Encoding ◽  
Content Type ◽  
Guide Rna ◽  
Eukaryotic Organisms

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii , failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii , we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10 9 cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii . The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.

The Chlamydomonas reinhardtii gtr Gene Encoding the Tetrapyrrole Biosynthetic Enzyme Glutamyl-tRNA Reductase: Structure of the Gene and Properties of the Expressed Enzyme

2005 ◽  
Vol 58 (5) ◽  
pp. 643-658 ◽  
Author(s):  
Alaka Srivastava ◽  
Vanessa Lake ◽  
Luiza A. Nogaj ◽  
Sandra M. Mayer ◽  
Robert D. Willows ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Biosynthetic Enzyme ◽  
Gene Encoding

scholarly journals Efficient Editing of the Nuclear APT Reporter Gene in Chlamydomonas reinhardtii via Expression of a CRISPR-Cas9 Module

2019 ◽  
Vol 20 (5) ◽  
pp. 1247 ◽  
Author(s):  
Daniel Guzmán-Zapata ◽  
José Sandoval-Vargas ◽  
Karla Macedo-Osorio ◽  
Edgar Salgado-Manjarrez ◽  
José Castrejón-Flores ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Transient Expression ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Model Organism ◽  
Nuclear Genome ◽  
Single Copy ◽  
Point Of View ◽  
Selection Marker ◽  
Gene Encoding

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.

scholarly journals A Low-CO2-Inducible Gene Encoding an Alanine:[alpha]-Ketoglutarate Aminotransferase in Chlamydomonas reinhardtii

1996 ◽  
Vol 112 (2) ◽  
pp. 677-684 ◽  
Author(s):  
Z. Y. Chen ◽  
M. D. Burow ◽  
C. B. Mason ◽  
J. V. Moroney
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Gene Encoding ◽  
Low Co2 ◽  
Inducible Gene

scholarly journals Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella

2007 ◽  
Vol 179 (3) ◽  
pp. 501-514 ◽  
Author(s):  
Kaiyao Huang ◽  
Dennis R. Diener ◽  
Aaron Mitchell ◽  
Gregory J. Pazour ◽  
George B. Witman ◽  
...  
Keyword(s):  
Rna Interference ◽  
Kidney Disease ◽  
Chlamydomonas Reinhardtii ◽  
Polycystic Kidney Disease ◽  
Fluorescence Recovery After Photobleaching ◽  
Intraflagellar Transport ◽  
Polycystic Kidney ◽  
Gene Encoding ◽  
Whole Cells ◽  
Flagellar Membrane

To analyze the function of ciliary polycystic kidney disease 2 (PKD2) and its relationship to intraflagellar transport (IFT), we cloned the gene encoding Chlamydomonas reinhardtii PKD2 (CrPKD2), a protein with the characteristics of PKD2 family members. Three forms of this protein (210, 120, and 90 kD) were detected in whole cells; the two smaller forms are cleavage products of the 210-kD protein and were the predominant forms in flagella. In cells expressing CrPKD2–GFP, about 10% of flagellar CrPKD2–GFP was observed moving in the flagellar membrane. When IFT was blocked, fluorescence recovery after photobleaching of flagellar CrPKD2–GFP was attenuated and CrPKD2 accumulated in the flagella. Flagellar CrPKD2 increased fourfold during gametogenesis, and several CrPKD2 RNA interference strains showed defects in flagella-dependent mating. These results suggest that the CrPKD2 cation channel is involved in coupling flagellar adhesion at the beginning of mating to the increase in flagellar calcium required for subsequent steps in mating.

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