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

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.

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

Eukaryotic Cell ◽

10.1128/ec.00213-14 ◽

2014 ◽

Vol 13 (11) ◽

pp. 1465-1469 ◽

Cited By ~ 156

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.

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The Circadian Clock of the Unicellular Eukaryotic Model Organism Chlamydomonas reinhardtii

Biological Chemistry ◽

10.1515/bc.2003.077 ◽

2003 ◽

Vol 384 (5) ◽

pp. 689-695 ◽

Cited By ~ 21

Author(s):

M. Mittag ◽

V. Wagner

Keyword(s):

Circadian Clock ◽

Chlamydomonas Reinhardtii ◽

Rna Binding ◽

Outer Space ◽

Expression Patterns ◽

Model Organism ◽

Nuclear Genome ◽

Circadian System ◽

Transcriptional Level ◽

Circadian Expression

Abstract The green unicellular alga Chlamydomonas reinhardtii, also called 'green yeast', emerged in the past years as a model organism for specific scientific questions such as chloroplast biogenesis and function, the composition of the flagella including its basal apparatus, or the mechanism of the circadian clock. Sequencing of its chloroplast and mitochondrial genomes have already been completed and a first draft of its nuclear genome has also been released recently. In C. reinhardtii several circadian rhythms are physiologically well characterized, and one of them has even been shown to operate in outer space. Circadian expression patterns of nuclear and plastid genes have been studied. The mode of regulation of these genes occurs at the transcriptional level, although there is also evidence for posttranscriptional control. A clock-controlled, phylogenetically conserved RNA-binding protein was characterized in this alga, which interacts with several mRNAs that all contain a common cis-acting motif. Its function within the circadian system is currently under investigation. This review summarizes the current state of the knowledge about the circadian system in C. reinhardtii and points out its potential for future studies.

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Rapid and high efficiency transformation of Chlamydomonas reinhardtii by square-wave electroporation

Bioscience Reports ◽

10.1042/bsr20181210 ◽

2019 ◽

Vol 39 (1) ◽

Cited By ~ 4

Author(s):

Liang Wang ◽

Lijing Yang ◽

Xin Wen ◽

Zhuoya Chen ◽

Qiaoying Liang ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Insertional Mutagenesis ◽

Molecular Mechanisms ◽

High Efficiency ◽

Model Organism ◽

Dependent Manner ◽

Exogenous Dna ◽

Square Wave ◽

Physiological Processes ◽

Final Yield

Abstract Chlamydomonas reinhardtii, the unicellular green algae, is the model organism for studies in various physiological processes and for bioindustrial applications. To explore the molecular mechanisms underlying physiological processes or to establish engineered cell lines, the exogenous DNA needs to be integrated into the genome for the insertional mutagenesis or transgene expression. However, the amount of selected marker DNA is not seriously considered in the existing electroporation methods for mutants library construction. Here, we reported a rapid-and-high-efficiency transformation technique for cell-walled strains using square-wave electroporation system. The final yield with this electroporation method was 2–6 × 103 transformants per μg exogenous DNA for cell-walled strains in a strain-dependent manner. In general, this electroporation technique was the easy and applicable way to build a mutant library for screening phenotypes of interest.

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High-efficiency retron-mediated single-stranded DNA production in plants

10.22541/au.163717150.01158451/v1 ◽

2021 ◽

Author(s):

Wenjun Jiang ◽

Gundra Sivakrishna Rao ◽

Rashid Aman ◽

Haroon Butt ◽

Radwa Kamel ◽

...

Keyword(s):

Mammalian Cells ◽

Genome Engineering ◽

High Efficiency ◽

Plant Biotechnology ◽

Fold Increase ◽

Gene Encoding ◽

Single Stranded Dna ◽

Homology Directed Repair ◽

Potential Applications

ABSTRACT Background: Retrons are a class of retroelements that produce multicopy single-stranded DNA (msDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the over-production of single-stranded DNA (ssDNA), genome engineering, and directed evolution in bacteria, yeast, and mammalian cells. However, no studies have shown retron-mediated ssDNA production in plants, which could unlock potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Main methods and major results: Here, we demonstrated retron-mediated over-production of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated CRISPR-retron-coupled ssDNA over-production and targeted HDR in N. benthamiana. Conclusion: We present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.

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Transient expression in mammalian cells of the bacterial reporter gene encoding mercuric reductase: effects of various regulatory elements

Gene ◽

10.1016/0378-1119(88)90055-8 ◽

1988 ◽

Vol 71 (2) ◽

pp. 381-390 ◽

Cited By ~ 2

Author(s):

Gerrit Veldhuisen ◽

Margriet Van Dijk ◽

Jorieke Meijer ◽

Betty E. Enger-Valk ◽

Peter H. Pouwels

Keyword(s):

Transient Expression ◽

Reporter Gene ◽

Mammalian Cells ◽

Regulatory Elements ◽

Gene Encoding ◽

Mercuric Reductase ◽

Expression In Mammalian Cells

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High efficiency gene transfer into mammalian cells

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1984.0137 ◽

1984 ◽

Vol 307 (1132) ◽

pp. 343-346 ◽

Cited By ~ 18

Keyword(s):

Gene Transfer ◽

Mammalian Cells ◽

High Efficiency ◽

Single Copy ◽

Rapid Screening ◽

Embryonic Cells ◽

Copy Gene ◽

Foreign Dna ◽

Foreign Genes ◽

Transient System

We have generalized the protocol of gene transfer, greatly increasing the variety of cells that can be used as recipients of foreign genes. Our approach has been to use a transient assay system that allows rapid screening of expression of foreign DNA. When the initial steps of gene transfer have been optimized with the transient system, these defined conditions are used to yield efficient stable transformation. We have seen that primate cells, including human cells, can be used in gene transfer experiments at levels sensitive enough to allow detection of single copy gene function. Recently we have also used this approach successfully with undifferentiated embryonic cells.

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I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1444 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1444-1448 ◽

Cited By ~ 71

Author(s):

Michel Cohen-Tannoudji ◽

Sylvie Robine ◽

André Choulika ◽

Daniel Pinto ◽

Fatima El Marjou ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Gene Targeting ◽

Mammalian Cells ◽

High Efficiency ◽

Shuttle Vector ◽

Embryonic Stem ◽

Gene Replacement ◽

Actin Binding ◽

Selection Marker

ABSTRACT Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 × 10−6) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.

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Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

Life Science Alliance ◽

10.26508/lsa.201900315 ◽

2019 ◽

Vol 2 (3) ◽

pp. e201900315

Author(s):

Stephan Eberhard ◽

Sona Valuchova ◽

Julie Ravat ◽

Jaroslav Fulneček ◽

Pascale Jolivet ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Replicative Senescence ◽

Model Organism ◽

Growth Conditions ◽

Telomeric Repeats ◽

Gene Encoding ◽

Unicellular Green Alga ◽

Chromosome Integrity ◽

Linear Chromosomes

Telomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. Expression of the reverse-transcriptase telomerase allows for extension of telomeric repeats to counteract natural telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly nondegenerate and that the telomeres form a protective structure, with a subset ending with a 3′ overhang and another subset presenting a blunt end. Although telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that telomeres shorten progressively in mutants of this gene. Telomerase mutants eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.

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Molecular characterization of Chlamydomonas reinhardtii telomeres and telomerase mutants

10.1101/519991 ◽

2019 ◽

Author(s):

Stephan Eberhard ◽

Sona Valuchova ◽

Julie Ravat ◽

Pascale Jolivet ◽

Sandrine Bujaldon ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Replicative Senescence ◽

Model Organism ◽

Growth Conditions ◽

Telomeric Repeats ◽

Gene Encoding ◽

Unicellular Green Alga ◽

Chromosome Integrity ◽

Linear Chromosomes

ABSTRACTTelomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. They shorten with each cell division because of the end-replication problem. Expression of the reverse transcriptase telomerase allows for extension of telomeric repeats to counteract telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly non-degenerate and that the telomeres form a protective structure, ending with a 3′ overhang. While telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that mutants of this gene display an “ever shortening telomere” phenotype and eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.

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