Transgenesis in microalga Chlamydomonas reinhardtii: current approaches

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INTRODUCTION

Eukaryotic microalgae are an extremely diverse group of photosynthetic unicellular microorganisms adapted to a wide range of ecological niches [1]. They account for about 50% of the total carbon fixation worldwide [2]. Due to their high metabolic plasticity, microalgae contain a wide range of beneficial substances such as natural antioxidants, vitamins, lipids, proteins, pigments, carbohydrates and secondary metabolites for pharmaceutical, agricultural, food and industrial applications. Microalgae are a rich source of pharmacologically active metabolites with antitumor, antibacterial, antifungal and antiviral properties. In addition to the production of endogenous compounds using bioengineering methods, microalgae can potentially be used as an inexpensive platform for the biosynthesis of various industrial enzymes and therapeutic proteins such as antibodies, hormones and vaccines [3]. Genetic engineering offers great prospects for the creation of highly productive strains of microalgae; being unicellular organisms, many species of microalgae remain haploid for most of their cell cycle, which reduces the time for obtaining and selecting of transformants. The use of microalgae as “cell factories” is a promising approach to produce commercially valuable compounds [4]. Some species of microalgae (e.g., Chlorella vulgaris Beijerinck and Chlamydomonas reinhardtii P.A. Dang.) have been granted GRAS (Generally Recognized as Safe) status — they are safe to eat [5].

Nevertheless, microalgae still remain relatively difficult targets for genetic manipulations [6]. The main problems are related to the need for a species-specific approach: cell transformation may be complicated by the peculiarities of membrane and cell wall structure in representatives of a particular taxon, and the developed transgenic construct may not be expressed due to a powerful gene silencing system. These difficulties explain the low efficiency of microalgae transformation and the scarce list of successfully edited species [6, 7].

C. reinhardtii (Chlamydomonas) is one of the most studied eukaryotic green algae, a popular model object for studying the genetics of photosynthesis, chloroplast biology, structure and function of sensory photoreceptors and photobehavior. Chlamydomonas is also a model organism for developing new approaches for genetic engineering of microalgae. The combination of rapid growth and ease of cultivation with the ability to photosynthesize, a well-studied life cycle, fully sequenced three genomes (nuclear, chloroplast, and mitochondrial) [8, 9], the possibility of transformation of each genome [10], and safety for humans make Chlamydomonas a valuable object for biotechnology. A great diversity of C. reinhardtii strains is maintained in various bioresource collections around the world. The only genetic collection in Russia is maintained at the Department of Genetics and Biotechnology of the St. Petersburg State University in Peterhof.

Currently, the Chlamydomonas chloroplast is successfully used as a platform for the biosynthesis of recombinant proteins with therapeutic properties (Table 1); for example, it can synthesize the birch pollen allergen Bet v 1 for allergy therapy and the spike protein of SARS-CoV-2 for vaccine development. The transformation techniques for C. reinhardtii plastome are well developed and allow to obtain stable high transgene expression [11].

 

Table 1. Therapeutic proteins synthesized in the chloroplast of C. reinhardtii

Таблица 1. Белки терапевтического назначения, синтезированные в хлоропласте C. reinhardtii

Purpose	Protein	Reference
Vaccine development	Foot-and-mouth disease virus VP1 protein	[12]
	E2 protein of classical swine fever virus (CSFV)	[13]
	p57 protein of the fish pathogen Renibacterium salmoninarum	[14]
	VP28 protein of shrimp white spot syndrome virus (WSSV)	[15]
	Fibronectin-binding domain D2 of Staphylococcus aureus	[16]
	acrV and vapA antigens of the fish pathogen Aeromonas salmonicida	[17]
	Pfs25, Pfs28, Pfs48/45, CelTOS antigens of Plasmodium falciparum	[18–21]
	Human papilloma virus 16 (HPV 16) protein E 7	[22, 23]
	MPT64 antigen of Mycobacterium tuberculosis	[24]
	Avian influenza virus (AIV) hemagglutinin H5	[25]
	p201 protein of ApoB100	[26]
	SARS-CoV-2 spike protein	[27]
Disease therapy	Vascular endothelial growth factor	[28]
	Interleukin 4	[29]
	TRAIL (TNFSF10)	[30]
	Human monoclonal antibodies	[28, 31–33]
	Human glutamate decarboxylase (hGAD65)	[34]
	Amphoterin (HMGB1)	[28]
	Immunotoxins	[35]
	Human somatotropin	[36]
	Cpl 1 and Pal endolysins of Streptococcus pneumoniae bacteriophage	[37]
	Interleukin 29	[38]
Allergy therapy	Peanut allergen Ara h 1	[39]
	Birch pollen allergen Bet v 1	[40]

 

Table 2. Main characteristics of the nuclear and chloroplast genomes of C. reinhardtii

Таблица 2. Основные характеристики ядерного и хлоропластного геномов C. reinhardtii

Characteristics	Genome
	nuclear	chloroplast
Size, base pairs	~110 × 106	~205 × 103
GC composition, %	~64	~36
Number of genes	~17585	99
Number of chromosomes	17	1
Number of copies in one cell	1	One chloroplast per cell, ~80 copies of the genome per chloroplast
Method of transgene integration	Random insertion, homologous recombination (by genetic editing)	Homologous recombination

 

Until recently, the introduction of transgenes into the nuclear genome of Chlamydomonas and their expression were associated with certain difficulties due to an efficient gene silencing system. Over the past few years, significant progress has been made in this area, including the use of novel CRISPR/Cas9-based techniques [41, 42].

This article presents an overview of current transgenesis techniques used for the unicellular green alga C. reinhardtii. The general design principles of transgenic constructs for transformation of nuclear and chloroplast genomes, selectable markers used to select transformants, techniques of transgene introduction (transformation) and genome editing using the CRISPR/Cas9 system are considered.

TRANSGENIC CONSTRUCTS DESIGN PRINCIPLES

A transgenic construct is a DNA cassette containing all the necessary regulatory elements for proper expression in the target organism. All constructs typically contain a promoter sequence, a gene of interest (transgene), a selectable marker and a terminator sequence. Depending on the need, the transgenic construct may carry additional elements to allow directed insertion into the genome or its controlled deletion from the genome.

The foreign gene can be integrated in the nuclear and chloroplast genome of C. reinhardtii. The main characteristics of each are presented in Table 2. In both cases, the efficient production of transformants requires adequate design of the transgenic construct, including codon optimization, selection of techniques for transgene introduction and selection of transformants, and methods for removal of marker sequences.

Transgenic constructs for transforming the nuclear and chloroplast genomes of C. reinhardtii differ significantly from each other (Fig. 1).

 

Fig. 1. Overall structure of transgenic constructs for transformation of the nuclear and chloroplast genome of C. reinhardtii: a — construct for transformation of the nuclear genome; b — construct for transformation of the chloroplast genome. ПГ — homology arms; П — promoter/5'-untranslated region; И — intron; T — terminator/3'-untranslated region; ПП — direct repeats. Additional explanations are given in the text

Рис. 1. Общий план строения трансгенных конструкций для трансформации ядерного и хлоропластного генома C. reinhardtii: a — конструкция для трансформации ядерного генома; b — конструкция для трансформации хлоропластного генома. ПГ — плечи гомологии; П — промотор/5'-нетранслируемая область; И — интрон; Т — терминатор/3'-нетранслируемая область; ПП — прямые повторы. Дополнительные пояснения к рисунку приведены в тексте

 

Until recently, transformation of the C. reinhardtii nuclear genome was associated with certain difficulties due to the effective silencing system [41]. In addition, before the advent of CRISPR/Cas technique, the transgenic construct could be integrated into the nuclear genome only by random insertion, which made the chloroplast of Chlamydomonas a more attractive platform, since transgene incorporation in it occurs only by homology. Several rules have been developed for the design of transgenes for their successful expression in the nuclear DNA of C. reinhardtii. First, it is necessary to use endogenous promoter (native or chimeric), terminator, 5'- and 3'-untranslated regions — the most frequently used are those from genes encoding the small subunit of ribulose bisphosphate carboxylase (rbcS2), heat shock protein hsp70A, and subunit II of the reaction center of photosystem I (psaD) [43]. Second, the codon composition of the transgene and the transforming genome should be taken into account [44]. Third, regular interruption of the transgene by introns [threefold repetition of intron 1 of rbcS2 (145 bp) in the transgene sequence and a single use of intron 2 of rbcS2 (329 bp) in the marker sequence] contributes to a high level of expression [45, 46]. This effect is achieved by a process called Intron-Mediated Enhancement (IME). The proposed mechanism is the interaction of RNA polymerase II with the spliceosome: if this interaction does not occur, RNA polymerase terminates transcription and the immature transcript is degraded. This phenomenon may underlie the ability to recognize the exogenous transcripts. The genes of C. reinhardtii are extremely rich in introns compared to other organisms: 92% of its genes contain introns with an average size of 373 bp. The average size of exons is 190 bp [41].

Directed insertion of a transgene into the nuclear genome of Chlamydomonas is possible only by homologous recombination along the homology arms (50 bp) through a reparation of double-stranded break induced by the CRISPR/Cas system [47]. The editing of the C. reinhardtii genome by CRISPR/Cas9 is discussed in detail below.

Transformation of the C. reinhardtii plastome is still more convenient and advantageous for the following reasons: 1) potential toxicity of the product to the cell is minimized, 2) targeted transgene integration by homologous recombination is possible without using CRISPR/Cas system, 3) high plastome ploidy and high level of chloroplast gene expression, 4) absence of a gene silencing system. One of the disadvantages of this approach is the lack of glycosylation of proteins synthesized in chloroplasts [11].

The transgenic construct for plastid transformation consists of a transgene and a selectable marker flanked by 100–200 bp homology arms, which are homologous to a selected insertion site in the host chloroplast DNA. For transgene and marker expression in the chloroplast of C. reinhardtii, the regulatory sequences of the nuclear gene rbcS2, chloroplast genes psaD, psaA, and psaB (photosystem I apoproteins), rbcL (large subunit of ribulose bisphosphate carboxylase), and psbA (core protein D1 of photosystem II) are most often used [11]. The GC composition of the transgene and chloroplast DNA must also be considered when designing a transgenic construct for Chlamydomonas chloroplast transformation.

The marker gene can be removed from the transformed genome using the Cre/loxP recombinase system [48]. For the plastome, the marker gene should be flanked on both sides by direct repeats. Removal of the selection factor during further propagation of the transformant can lead to internal homologous recombination between repeats with removal of the selectable marker from the chloroplast genome [49].

SELECTABLE MARKERS

Selectable markers are used to create conditions for selection of transformed cells. The marker sequences most widely used in C. reinhardtii transformation are presented in Table 3.

The first selectable markers for nuclear genome transformation used in C. reinhardtii were its own genes that restore metabolic pathways in strains carrying mutations in the ARG7 and NIT1 genes. Auxotrophic mutants transformed with normal copies of these genes are selected as prototrophs. This “marker-free” approach based on the compensation of the mutation by a wild-type gene copy remains popular and is widely used in the transformation of nuclear and chloroplast DNA in the corresponding mutants [58, 59].

 

Table 3. The most widely used selection markers for the transformation of nuclear and chloroplast DNA of C. reinhardtii

Таблица 3. Наиболее широко используемые при трансформации ядерной и хлоропластной ДНК C. reinhardtii селекционные маркеры

Target	Gene	Product	Selection	Reference
Nuclear DNA	ARG7	Argininosuccinate lyase	Arginine-free medium	[50]
	NIT1	Nitrate reductase	Nitrate-free medium	[51]
	CRY1-1	Mutant ribosomal protein S14/rp59	Medium with emetine, cryptopleurin	[52]
	aph7''	Aminoglycoside phosphotransferase	Medium with hygromycin B	[43]
	aphVIII	Aminoglycoside phosphotransferase	Medium with paromomycin, neomycin, kanamycin	[53]
	ble	Bleomycin-binding protein	Fluomycin medium	[44, 54]
Nuclear and chloroplast DNA	aadA	Aminoglycoside adenylyltransferase	Medium with spectinomycin, streptomycin	[55, 56]
Chloroplast DNA	aphA6	Aminoglycoside phosphotransferase	Medium with kanamycin, amikacin	[57]

 

The ARG7 gene encodes argininosuccinate lyase and restores the metabolic pathway of arginine biosynthesis during successful transformation of an arginine-dependent C. reinhardtii mutant strain, allowing the transformants to grow on minimal medium without arginine supplementation.

The NIT1 gene encodes nitrate reductase, an enzyme that allows the successfully transformed C. reinhardtii mutant strain to utilize nitrate as the sole source of nitrogen in the medium.

A mutant strain of C. reinhardtii resistant to the translation inhibitors, emetin and cryptopleurin, was isolated and studied in 1994 [52]. This resistance attributed to a missense mutation (CRY1-1) in the CRY1 gene. The sequence of the mutant CRY1-1 gene under the control of the endogenous rbcS2 promoter can be used for transformation of wild-type strains of C. reinhardtii.

The use of exogenous selectable markers has simplified the transformant selection process and eliminated the necessity of using only C. reinhardtii mutants as transgene acceptors. Currently, the most effective and widely used exogenous selectable markers are various antibiotic resistance genes.

The aph7'' gene of Streptomyces hygroscopicus encodes a phosphotransferase that deactivates the antibiotic hygromycin B. A chimeric construct including the aph7'' gene sequence of S. hygroscopicus, β2-tubulin gene promoter, and first intron with the 3'-untranslated region of rbcS2 gene of C. reinhardtii is used for Chlamydomonas transformation [43].

The aphVIII gene of Streptomyces rimosus encodes a phosphotransferase that deactivates the antibiotics paromomycin, neomycin, and kanamycin. The highest transformation efficiency was achieved using a chimeric construct with the sequences of the 5'-untranslated region of the heat shock protein hsp70A gene of C. reinhardtii, first intron of the rbcS2 gene of C. reinhardtii, the aphVIII gene of S. rimosus, and the 3'-untranslated region of the rbcS2 gene of C. reinhardtii [53].

The ble gene of Streptoalloteichus hindustanus encodes an enzyme that binds the antibiotic phleomycin. A chimeric construct with this gene, including sequences of the first intron and the 5'- and 3'-untranslated regions of the rbcS2 gene of C. reinhardtii, showed consistently high transformation and expression efficiency [44, 54].

The aadA gene of Escherichia coli encodes an enzyme that confers resistance to the antibiotics spectinomycin and streptomycin. For transformation of Chlamydomonas plastome, a construct with the sequences of the promoter of the chloroplast atpA gene and the 3'-untranslated region of rbcL gene of C. reinhardtii is used [55]. To transform nuclear DNA, the aadA gene of E. coli is combined with the 5'- and 3'-untranslated regions of the rbcS2 gene of C. reinhardtii [56].

The aphA6 gene of Acinetobacter baumannii encodes a phosphotransferase that deactivates the antibiotics kanamycin and amikacin. The highest expression of this gene in the chloroplast of Chlamydomonas was observed using a compact chimeric construct with the sequence of the 5'-untranslated region of the psbA gene of C. reinhardtii, the aphA6 gene of A. baumannii, and the 3'-untranslated region of the rbcL gene of C. reinhardtii [57].

Fluorescent proteins, such as mCherry [60] and mVenus [45], can also be used as a selectable markers. Transformants are screened using a confocal microscope.

CHLAMYDOMONAS MODULAR CLONING KIT

Rapid and predictable modification of the C. reinhardtii genome can be achieved using standardized tools and techniques. For this purpose, a set of tools for modular cloning based on Golden Gate technology — Chlamydomonas Modular Cloning kit was developed [61]. The system uses BsaI and BpiI restrictases and consists of three sets of vectors for cloning (levels 0, 1, and 2). Level 0 vectors contain individual elements such as promoters, 5'- and 3'-untranslated regions, coding sequences, terminators, selectable markers and introns. These modules are then assembling into one complete transcriptional unit within the level 1 vector during the restriction/ligation reaction. In the second step, several transcriptional units from level 1 vectors can be combined within a level 2 vector to create multigenic clusters encoding enzymes of new metabolic pathways [42].

A ready-to-use standardized toolkit allows rapid (in 4–5 weeks) creation of modified cells for basic research and biotechnology.

C. REINHARDTII TRANSFORMATION METHODS

Transformation is the process of integrating a functional DNA fragment — a transgenic construct — into target cells. Currently, three methods of transgene delivery into Chlamydomonas cells are widely used: glass beads, electroporation, and biolistic [10].

Glass beads are the most affordable, simple, and efficient method of nuclear [62] and plastome [63] genome transformation. The transgene, C. reinhardtii cells without cell wall (using autolysin or a cell wall-less mutant strain), and glass beads are mixed on a vortex. The glass beads rupture the membrane when shaken, allowing the transfer of exogenous DNA into the cell. They can also be used to deliver ribonucleoprotein (RNP) complexes formed by Cas nuclease and guide RNA (gRNA) into cells.

The electroporation method, first used on Chlamydomonas in 1991 [64], involves treating cells with high-voltage pulses on an electroporator, which forms pores with a diameter of 20–40 nm in the membrane, through which foreign DNA enters the cell. The method allows achieving a high yield of transformants, but requires adaptation of conditions depending on the electroporator and the specific strain of C. reinhardtii used [65]. Electroporation is performed by generation of exponential or square wave pulses. Square waves are more controllable and lead to the generation of a greater number of transformants [66]. In addition, square wave pulses may be used for transformation of cell-walled C. reinhardtii cells without its removal by autolysin [67], while exponential wave pulses are suitable only for cells lacking a cell wall [65]. Experiments on electroporation of cell-walled C. reinhardtii cells by exponential waves on the electroporator BTX ECM 600 without autolysin treatment resulted in transformation frequency of 3%, and with autolysin treatment — in 87%, confirming and supplementing the previously obtained data [47]. By optimizing parameters such as transgene concentration, wave type, and voltage, the transformation rate by electroporation can be higher than that of the glass bead method [68]. The overall efficiency of transformation is significantly increased by using the latest models of electroporators, such as the NEPA21. This device sequentially generates three types of square wave pulses: “poring pulse” with high voltage and short pulse length, creating pores in the cell membrane, series of “transfer pulses” with low voltage and long pulse length, and “transfer pulse” with reversed polarity for efficient delivery of DNA molecules into cells [67]. Electroporation is a standard method for C. reinhardtii transformation, especially for delivering the Cas/gRNA RNP complexes into cells.

The biolistic method involves bombarding recipient cells with heavy metal particles (mainly gold or silver) coated with transgenic DNA in a vacuum chamber. This method can be used to transform the DNA of both the cell nucleus and organoids, such as mitochondria and plastids. Currently, this technique is mainly used for transforming Chlamydomonas chloroplast.

Other methods of Chlamydomonas transformation, such as Agrobacterium-mediated transformation, have been developed and tested, but they have not been widely used [10]. This approach is much more complex and yet has no significant advantages over electroporation [69].

Recently, a novel system for the delivery of Cas9/gRNA RNP complexes into C. reinhardtii cells was developed using the cell-penetrating peptide pVEC (amino acid sequence: LLIILRRRRRIRKQAHAHSK). It can be used as an alternative to electroporation in terms of availability and efficiency [70].

GENOME EDITING USING CRISPR/CAS9 IN C. REINHARDTII

CRISPR/Cas9 is a system of Cas9 protein with nuclease activity and a non-coding gRNA complementary to DNA in the target site. The CRISPR/Cas9 genome editing system generally functions as follows: the assembled Cas9/gRNA complex scans the DNA sequence for a match of the gRNA spacer sequence to the target site (protospacer) on the DNA and the presence of the protospacer adjacent motif (PAM) sequence nearby. After formation of the gRNA–DNA complex, the Cas9 endonuclease makes a double-stranded break 3–4 nucleotides upstream of the PAM. A transgenic construct carrying homology arms to the restriction site can be targeted into the resulting in vivo gap (homologous recombination) [71].

 

Fig. 2. Strategies for delivering components of the CRISPR/Cas9 system into C. reinhardtii cells: a — integrative system; b — episomal system; c — ribonucleoprotein system. Additional explanations are given in the text

Рис. 2. Стратегии доставки компонентов системы CRISPR/Cas9 в клетки C. reinhardtii: a — интегративная система; b — эписомная система; c — рибонуклеопротеиновая система. Дополнительные пояснения к рисунку приведены в тексте

 

Novel methods of Chlamydomonas transformation are based on the CRISPR/Cas9 technology. The components of the system can be delivered into the cell by the following ways: 1) plasmid vectors carrying sequences encoding Cas9 protein and gRNA; 2) in vitro assembled Cas9/gRNA RNP complex (Fig. 2). The use of the RNP complex has several advantages including avoidance of random insertional mutagenesis and reduction of off-target editing probability, consequently demonstrating higher efficiency.

The integrative strategy uses one or two plasmids encoding the Cas9 and gRNA genes. Once introduced into the cell, the plasmid is linearized by cellular restriction enzymes and integrated into a random location in the C. reinhardtii genome. Upon its expression, the Cas9/gRNA complex is assembled, which produces a double-stranded break in the target site [72]. The first attempt to produce Cas9 endonuclease and gRNA from the same plasmid in C. reinhardtii cells yielded a single colony of transformants for the FKB12 gene from >10⁹ cells taken [73]. Co-transfection of cells with two independent plasmids resulted in APT gene knockout lines with an efficiency of 3–30% [74].

A method was developed to prevent integration of Cas9 and gRNA gene sequences into the genome. It was shown that plasmids containing the yeast centromeric sequence, replication initiation site and yeast selectable marker (CEN6-ARSH4-HIS3) replicate autonomously in cells and are not incorporated into the genome of the diatoms Thalassiosira pseudonana Cleve and Phaeodactylum tricornutum Bohlin [75, 76]. Experiments with this technique were continued in 2019 and stable transformants of the green microalgae Acutodesmus obliquus (Turpin) Hegewald & Hanagata and Neochloris oleoabundans S. were obtained. However, later the integration of the episomal plasmid into the nuclear genome occurred [77]. In another study [78], the nitrate reductase gene in the microalgae Nannochloropsis oceanica Suda & Miyashita was successfully knocked out using an episomal vector. The episomal plasmid was eliminated from the transformant cells after approximately 30 cell division cycles, yielding a nontransgenic line. The use of an episomal plasmid has not yet been tested for transformation of C. reinhardtii. Nevertheless, the development of such plasmid is easy, and the possibility of constructing nontransgenic lines without markers makes this method comparable in its applicability to the RNP system [72].

As the expression of foreign genes in C. reinhardtii is often inefficient, especially if they encode large proteins, such as Cas9 [60, 73], the most commonly used delivery method involves in vitro preassembly of the Cas9/gRNA RNP complex, which is directly used for transformation. The RNP complex rapidly degrades in the cell, which limits endonuclease activity and minimizes the risks of off-target effects, compared to constitutive expression of Cas9 [79].

The CRISPR/Cas9 technique can be used for targeted gene knockout and knockin in Chlamydomonas. The components of the system are delivered into the cell together with donor DNA cassette (transgene construct), which can be incorporated into the double-stranded break site. Addition of homology arms flanking the restriction site of Cas9 nuclease to the donor DNA sequence are required for homologous recombination, providing controllability and predetermination of the result. This method increases the yield of transformants up to 85% [47, 60].

The general protocol for C. reinhardtii transformation was significantly refined in 2017. According to observations, heat shock treatment of cells prior to transformation is a critical parameter to maximize the number of transformants obtained. It is assumed that some physiological changes favorable for the activity of the Cas9/gRNA complex and/or for the processes associated with DNA repair occur in cells. The optimal parameters for post-transformation cell recovery have also been determined [60].

In 2020, a highly efficient method of targeted insertional mutagenesis (TIM) was developed. The authors conducted a series of paired experiments and identified parameters crucial for high efficiency of Chlamydomonas transgenesis based on the CRISPR/Cas RNP strategy. Optimization of the nuclear genome transformation protocol allowed them to achieve mutagenesis efficiency from 40 to 95% in separate experiments, even with multiplex editing [47].

In 2023, a protocol was developed for the routine generation of mutant Chlamydomonas lines without residual selectable markers [80]. In the same year, using the CRISPR/Cas RNP strategy, transformants expressing an optimized bacterial phytase transgene embedded in the second exon of the nuclear gene NIT1 and stable for 100 days of cultivation were obtained for the first time. Notably, cells transformed by conventional methods (without using the RNP complex or CRISPR/Cas method) lost the transgene after two generations (about 20 days of cultivation) [81].

Many attempts were made in order to optimize conditions for Chlamydomonas transformation and increase the efficiency of gene editing. These attempts involved evaluation of various factors such as growth conditions and cell preparation methods, required density of the cell culture, concentration of the Cas9/gRNA RNP complex, technique of cell wall removal, selection and optimization of transformation methods for various Chlamydomonas strains and equipment, and techniques for recovery and selection of transformants. The protocol for nuclear genome transformation of the microalgae C. reinhardtii via CRISPR/Cas RNP complex is summarized below.

The development of advanced gene editing techniques has played an important role in the establishment of C. reinhardtii as a model organism for the study of microalgae. Achievements of recent years, including the development of transgene design rules, creation of a

 

1. Cell cultivation and preparation
Grow cells on solid TAP medium (1.5% agar, arginine, yeast autolysate) at 20°C–25°C in a 14 h light (200–300 µE/m2 × s):10 h dark cycle
Harvest cells on the third day (exponential growth phase), resuspend in sterile water and centrifuge at 5,000 rpm for 5 min
Resuspend the cells of cell-walled strains in autolysin to a cell density of 1 × 108 cells/mL and incubate at room temperature for 30 min	Resuspend the cells of cell wall-less strains in liquid TAP + 40 mM sucrose medium to a cell density of 1 × 108 cells/mL
2. HEAT SHOCK
Centrifuge cell suspension at 5,000 rpm for 5 min
Resuspend cell pellet in liquid TAP + 40 mM sucrose and centrifuge at 5,000 rpm for 5 min. Resuspend cell pellet in liquid TAP + 40 mM sucrose to a cell density of 1 × 108cells/mL
3. Transformation
Glass beads	Electroporation (BioRad GenePulser Xcell electroporator)
Mix 300 μL of resuspended cells (3 × 107 cells/mL) with 5 μL of Cas9/gRNA RNP complex (3 μM Cas9:gRNA) and 1 μg of transgenic construct in a tube with 0.45 nm sterile glass beads (500–600 μg)	For one 2 mm cuvette mix 40 μL of resuspended cells (4 × 106 cells/mL) with 4 μL of Cas9/gRNA RNP complex (3 μM Cas9:gRNA) and 1 μg of the transgenic construct
Vortex at top speed for 15 sec, rest for 10 sec, vortex again for 10 sec	Electroporate in 2 mm cuvette at square wave, 300 V, 6 pulses, pulse length 4 ms, pulse interval 100 ms. The cuvette with the mixture should be shortly cooled in ice before and after electroporation
Resuspend each reaction mixture in 600 µL of fresh liquid TAP + arginine medium in a separate tube
4. Cell recovery and selection
Cultivate cell suspension at 20°C–25°C under dim light for 24 h
Vortex the culture mixture and pipette onto plates with solid TAP selective medium (1.5% agar, 10 µg/mL antibiotic)
Grow cells at 20°C–25°C in a 14 h light (200–300 µE/m2 × s):10 h dark cycle until the transformant colonies formation (1–2 weeks)

 

Chlamydomonas Modular Cloning kit, and adaptation of the CRISPR/Cas9 system for targeted genome editing, have significantly increased the efficiency of transgene expression in C. reinhardtii cells and the overall potential of microalgae genetic engineering. This opens prospects for the creation of new producer strains, valuable for both fundamental research and practical use, and will make it possible to replenish the resources of the Peterhof genetic collection of C. reinhardtii strains, which is the only one in Russia.

CONCLUSION

The unicellular green alga C. reinhardtii is a well-established model organism in molecular biology and plant genetics. The degree of study of genetic systems of Chlamydomonas is a standard for other microalgae. The availability, ease of cultivation, and the possibility of manipulation of all three genomes (nuclear, chloroplast, and mitochondrial) have allowed to make significant contributions to the development of new approaches for genetic engineering in microalgae.

The CRISPR/Cas9 genome editing system has opened up a new chapter in C. reinhardtii genetic engineering by enabling directed transgene insertion into the nuclear genome. In recent years, significant advances have been made in the development of highly efficient Chlamydomonas transformation protocols and the production of valuable enzymes and proteins. The Chlamydomonas Modular Cloning kit, introduced in 2018, aims to unify the design process for transgenic constructs. Standardized genetic elements can be readily substituted and assembled into complete expression units, which further enable the creation of new multigene constructs. This opens up the possibility of introducing new metabolic pathways into algae cells [42, 61].

Microalgae are an interesting object for introduction and adaptation of new approaches to genome engineering. They contain a wide range of useful substances: antioxidants, lipids, proteins, carbohydrates and secondary metabolites. For example, the green alga Chlorella vulgaris can be used as a producer of 18 amino acids, 20 vitamins, and many minerals [82]. Microalgae are an exceptionally rich source of pharmacologically active metabolites with antitumor, antibacterial, antifungal, and antiviral properties, and are also suitable for wastewater treatment and biofuel production.

Currently, heterotrophic platforms are usually used for biosynthesis of valuable compounds: bacteria, yeasts, cell cultures. Nevertheless, the development of bioeconomy and the need for sustainable alternatives to petrochemical products stimulate interest in the search and use of new producers, such as photosynthetic microalgae. Advances in the biosynthesis of recombinant proteins in C. reinhardtii chloroplasts along with the ever-improving transformation techniques have indicated the high commercial potential of this alga [36]. There are a number of specific properties that make microalgae ideal candidates for the production of various food additives, commercial enzymes, and therapeutic proteins: 1) affordable cultivation, fast biomass build-up, and easily controlled and scalable; 2) quick production of transgenic microalgae in 4–5 weeks; 3) the ability to transform the plastome and nuclear genome allows the production of multiple transgenic proteins (or an entire protein complex) in a single organism. Although bacterial and yeast systems are more economical, they have several disadvantages. It is not always possible to obtain functional proteins due to specific features in the post-translational modifications and protein folding. The use of another photosynthetic alternative platform — terrestrial plants — for large scale cultivation is economically not viable as it poses economic burden along with the long time required to obtain transgenic lines (4–6 years), the possibility of gene flow through pollen into the environment, and the ban on open cultivation of transgenic plants [4, 31, 83].

There is every reason to believe that microalgae in the future may become an alternative platform for the development of economically valuable compounds in industrial and medical biotechnology. As the most studied microalga, C. reinhardtii has the genetic potential to become a major platform in the microalgae biotechnology [84].

ADDITIONAL INFORMATION

Authors’ contribution. All authors have made a significant contribution to the development of the concept and preparation of the article, as well as read and approved the final version before its publication. Personal contribution of the authors: P.A. Virolainen — search and analysis of literature, writing the main part of the text; E.M. Chekunova — development of the concept, making final edits.

Funding source. The authors acknowledge Saint Petersburg State University for a research grant (project ID Pure 115624290).

Competing interests. The authors declare that they have no competing interests.

About the authors

Pavel A. Virolainen

Saint Petersburg State University

Email: st085618@student.spbu.ru
ORCID iD: 0000-0001-5918-9395
SPIN-code: 6564-9350
Scopus Author ID: 57883811500

PhD student

Russian Federation, 7/9 Universitetskaya emb., Saint Petersburg, 199034

Elena M. Chekunova

Saint Petersburg State University

Author for correspondence.
Email: e.chekunova@spbu.ru
ORCID iD: 0000-0001-8942-4771
SPIN-code: 2788-6386
Scopus Author ID: 6701797455

Dr. Sci. (Biology), Senior Teacher

Russian Federation, 7/9 Universitetskaya emb., Saint Petersburg, 199034

References

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