There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H2 yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H2 production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.

Genetic Engineering of Algae for Enhanced Biofuel Production

http://www.genetics.wustl.edu/sdlab/PDFs/Li2004.pdf

Comparative Genomics Identifies a Flagellar and Basal Body Proteome that Includes the BBS5 Human Disease Gene

The unicellular green alga Chlamydomonas offers a simple life cycle, easy isolation of mutants, and a growing array of tools and techniques for molecular genetic studies. Among the principal areas of current investigation using this model system are flagellar structure and function, genetics of basal bodies (centrioles), chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. A genome project has begun with compilation of expressed sequence tag data and gene expression studies and will lead to a complete genome sequence. Resources available to the research community include wild-type and mutant strains, plasmid constructs for transformation studies, and a comprehensive on-line database.

Chlamydomonas as a model organism

Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, user-provided DNA can be used as a template for repair by homologous recombination (HR), leading to the introduction of desired sequence changes. This review describes three classes of targetable cleavage reagents: zinc-finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nucleases (RGNs). As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. This review discusses the properties, advantages, and limitations of each system, as well as the specific considerations required for t...

https://www.annualreviews.org/doi/pdf/10.1146/annurev-biochem-060713-035418

Genome Engineering with Targetable Nucleases

Engineering algae for biohydrogen and biofuel production

A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii.

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.

Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9.

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant (Pm-R) clones with expressing nucleases. Of these Pm-R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.12066

Nuclear gene targeting in Chlamydomonas using engineered zinc‐finger nucleases

Homologous DNA recombination (HR) allows the deletion (knockout), repair (rescuing), and modification of a selected gene, thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an inefficient process in most eukaryotes, including algae, plants, and animals, due to the dominance of integration of the applied DNA into nonhomologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3-phosphotransferase gene, aphVIII, that single-stranded DNA can recombine with a homologous endogenous DNA region of interest. Nonhomologous DNA integration appeared to be more than 100-fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes. Green microalgae are of great value both as organisms for fundamental biological research and as a resource for biotechnological industry. Because of its well-defined genetics Chlamydomonas reinhardtii is an ideal system for studying photosynthesis, chloroplast biogenesis, flagellar function, phototaxis, etc. However, genetic and molecular analyses of nuclear transformants reveal that integration of the DNA occurs predominantly via nonhomologous recombination (NHR) resulting in the introduction of the marker DNA at apparently random loci (7). Further application of C. reinhardtii as a model system and for technical use urgently demands targeted gene disruption and gene replacement. Use of targeted gene disruption. Targeted gene disruption allows the introduction of in vitro-generated mutations, including null mutations, into the genome of an organism. Homologous gene replacement has significant advantages over random integration (see Fig. S1 in the supplemental material) because it does not have drawbacks such as gene dosage effects, position effects, and the necessity of detection against a background of endogenous gene activity. The successful application of targeted gene disruption and replacement is dependent on the ratio of homologous to illegitimate recombination events during integrative transformation. This ratio is extremely variable among different eukaryotes. In several lower eukaryotes such as yeasts (12), some filamentous fungi (8), Trypanosomatideae (6), and the moss Physcomitrella patents (a plant with the predominance of the haplophase in the life cycle) (28) have a ratio of homologous to random integration above 10%. In archaea, in many lower eukaryotes like algae, and especially in most higher eukaryotes, the ratio of homologous to nonhomologous recombination events is very low. It varies between 10 2 and 10 3 in animal cells (4) and between 10 3 and 10 6

Irina Sizova

Papers

A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii.

Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9.

Nuclear gene targeting in Chlamydomonas using engineered zinc‐finger nucleases

Nuclear-Gene Targeting by Using Single-Stranded DNA Avoids Illegitimate DNA Integration in Chlamydomonas reinhardtii†

Nuclear gene targeting in Chlamydomonas as exemplified by disruption of the PHOT gene.