SAGA1 and MITH1 produce matrix-traversing membranes in the CO-fixing pyrenoid.

Approximately one-third of global CO assimilation is performed by the pyrenoid, a liquid-like organelle found in most algae and some plants. Specialized pyrenoid-traversing membranes are hypothesized to drive CO assimilation in the pyrenoid by delivering concentrated CO, but how these membranes are made to traverse the pyrenoid matrix remains unknown. Here we show that proteins SAGA1 and MITH1 cause membranes to traverse the pyrenoid matrix in the model alga Chlamydomonas reinhardtii.

Biogenesis, engineering and function of membranes in the CO -fixing pyrenoid.

Approximately one-third of global CO assimilation is performed by the pyrenoid , a liquid-like organelle found in most algae and some plants . Specialized membranes are hypothesized to drive CO assimilation in the pyrenoid by delivering concentrated CO , but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant .

Multi-omics analysis of green lineage osmotic stress pathways unveils crucial roles of different cellular compartments.

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages.

Compact zinc finger architecture utilizing toxin-derived cytidine deaminases for highly efficient base editing in human cells.

Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddA, that can act upon double-stranded DNA.

Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis.

Oxygen (O), a dominant element in the atmosphere and essential for most life on Earth, is produced by the photosynthetic oxidation of water. However, metabolic activity can cause accumulation of reactive O species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O sensitivity screen on a genome-wide insertional mutant library of the unicellular alga .

Systematic characterization of gene function in the photosynthetic alga Chlamydomonas reinhardtii.

Most genes in photosynthetic organisms remain functionally uncharacterized. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. A total of 59% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes.

Enhancing gene editing specificity by attenuating DNA cleavage kinetics.

Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites.

A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii.

Development of Bag-1L as a therapeutic target in androgen receptor-dependent prostate cancer.

Targeting the activation function-1 (AF-1) domain located in the N-terminus of the androgen receptor (AR) is an attractive therapeutic alternative to the current approaches to inhibit AR action in prostate cancer (PCa). Here we show that the AR AF-1 is bound by the cochaperone Bag-1L. Mutations in the AR interaction domain or loss of Bag-1L abrogate AR signaling and reduce PCa growth.

Endogenous sequence patterns predispose the repair modes of CRISPR/Cas9-induced DNA double-stranded breaks in Arabidopsis thaliana.

The possibility to predict the outcome of targeted DNA double-stranded break (DSB) repair would be desirable for genome editing. Furthermore the consequences of mis-repair of potentially cell-lethal DSBs and the underlying pathways are not yet fully understood. Here we study the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced mutation spectra at three selected endogenous loci in Arabidopsis thaliana by deep sequencing of long amplicon libraries.

CRISPR/Cas-Mediated In Planta Gene Targeting.

The recent emergence of the CRISPR/Cas system has boosted the possibilities for precise genome engineering approaches throughout all kingdoms of life. The most common application for plants is targeted mutagenesis, whereby a Cas9-mediated DNA double-strand break (DSB) is repaired by mutagenic nonhomologous end joining (NHEJ). However, the site-specific alteration of a genomic sequence or integration of a transgene relies on the precise repair by homologous recombination (HR) using a suitable donor sequence: this poses a particular challenge in plants, as NHEJ is the preferred repair mechanism for DSBs in somatic tissue.

CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases.

The CRISPR/Cas system has recently become the most important tool for genome engineering due to its simple architecture that allows for rapidly changing the target sequence and its applicability to organisms throughout all kingdoms of life. The need for an easy-to-use and reliable nuclease is especially high in plant research, as precise genome modifications are almost impossible to achieve by Agrobacterium-mediated transformation and the regeneration of plants from protoplast cultures is very labor intensive. Here, we describe the application of the Cas9 nuclease to Arabidopsis thaliana for the induction of heritable targeted mutations, which may also be used for other plant species.

Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes.

Duplication of existing sequences is a major mechanism of genome evolution. It has been previously shown that duplications can occur by replication slippage, unequal sister chromatid exchange, homologous recombination, and aberrant double-strand break-induced synthesis-dependent strand annealing reactions. In a recent study, the abundant presence of short direct repeats was documented by comparative bioinformatics analysis of different rice genomes, and the hypothesis was put forward that such duplications might arise due to the concerted repair of adjacent single-strand breaks (SSBs).

Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus.

The application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system of Streptococcus pyogenes (SpCas9) is currently revolutionizing genome engineering in plants. However, synthetic plant biology will require more complex manipulations of genomes and transcriptomes. The simultaneous addressing of different specific genomic sites with independent enzyme activities within the same cell is a key to this issue.

The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny.

The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double-strand breaks (DSBs) at pre-selected genomic sites that are repaired by non-homologous end joining (NHEJ) in an error-prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs.

Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.

Engineered nucleases can be used to induce site-specific double-strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end-joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ.

DNA recombination in somatic plant cells: mechanisms and evolutionary consequences.

In somatic cells, recombination is a means of DNA damage repair. The most severe type of damage in nuclear DNA is double-strand breaks (DSBs) which may be repaired via either non-homologous end joining (NHEJ) or homologous recombination (HR). In this review, we will summarize the basic features, the mechanisms, and the key players of both repair modes in plants with a focus on the model plant Arabidopsis thaliana.

Gene targeting in plants: 25 years later.

Only five years after the initiation of transgenic research in plants, gene targeting (GT) was achieved for the first time in tobacco. Unfortunately, the frequency of targeted integration via homologous recombination (HR) was so low in comparison to random integration that GT could not be established as a feasible technique in higher plants. It took another 25 years and great effort to develop the knowledge and tools necessary to overcome this challenge, at least for some plant species.

Synthetic nucleases for genome engineering in plants: prospects for a bright future.

By inducing double-strand breaks (DSB), it is possible to initiate DNA recombination. For a long time, it was not possible to use DSB induction for efficient genome engineering due to the lack of a means to target DSBs to specific sites. This limitation was overcome by development of modified meganucleases and synthetic DNA-binding domains.

In planta gene targeting.

The development of designed site-specific endonucleases boosted the establishment of gene targeting (GT) techniques in a row of different species. However, the methods described in plants require a highly efficient transformation and regeneration procedure and, therefore, can be applied to very few species. Here, we describe a highly efficient GT system that is suitable for all transformable plants regardless of transformation efficiency.