This year, we use transcriptome analysis and CRISPR/Cas9 system to discover and manipulate lipid metabolism-related genes in our chassis, Chlamydomonas reinhardtii to enrich lipid productivity and maintain culture efficiency. During the process, we are surprised to find that throughout the entire iGEM competition few teams have successfully used CRISPR technology on algae engineering. Actually, we are the second team to try to construct a CRISPR system for algae not to mention successfully completing the process.

On this page, we clearly introduce how we innovatively construct a CRISPR/Cas9 system that is especially for Chlamydomonas reinhardtii in great detail. Not only have we successfully proved the feasibility of the genome editing tool in algae, but we also bring out omics research that assists researchers to discover target genes and construct a CRISPR off-target model to help search for a suitable gRNA.

Microalgae are precious research and application materials that have been proven to have functions such as producing biofuels, fixing carbon dioxide, purifying water bodies, etc. Synthetic biology on microalgae has been a hot topic for many years so as to improve their functions to better fulfil humanity’s needs. Locating, understanding and manipulating desired genes are crucial in the process. In Chlipid, our team desire to employ the powerful genome editing tool CRISPR in algal research which few iGEM teams have tried before.

We designed the CRISPR/Cas9 system for editing Chlamydomonas reinhardtii based on the results of Greiner et al. In this system, we selected mCherry which is the best available reporter gene in the plant field, and StayGold, a bright green fluorescent protein that has recently been shown to be highly photostable. And then we optimized the two reporters according to the codon preference of Chlamydomonas reinhardtii. At the same time, each vector carried an optional marker box, C. reinhardtii which could be granted resistance to Hyg. In the selection of promoters, we selected RBCS2 and HSP70A. For terminators, polyT Term and RBCS2 Term were selected.

To prove our CRISPR system design, we performed the following tests that clearly indicated the CRISPR vectors we designed could be successfully transformed and expressed normally in Chlamydomonas reinhardtii (Stain CC-503) which suggests we have constructed a CRISPR system for algae successfully, a huge breakthrough in the history of iGEM. (More information on https://2022.igem.wiki/uestc-biotech/results)

We used Golden Gate Assembly to construct each element into pTX2038 and pTX2040 vectors(Figure 1A and 1B), and amplified and expressed them in E. coli DH-5α to obtain the basic skeleton vector. To verify the successful construction of the vector, PCR amplification was performed.


The brightness of the primer fragment amplified PCR product matched with the DNA Marker marker position (Figure 1C), which initially proved that the sgRNA of the target gene was inserted into the vector. Subsequently, we performed Sanger forward sequencing and sequence comparison for each insert site to further verify the successful vector construction.

We chose to use the method of electro-transformation to transform Chlamydomonas reinhardtii. In order to improve the efficiency of electric electro-transformation, we used electroporation buffer (ME Suc): Use convert reagent with MAX efficiency ™ (Therfisher, # A24229) to carry out the experiment.
The number of monoclone which had been successfully transferred into pTX2038 and pTX2040 in the plate was counted respectively and their transformation efficiencies were also calculated respectively (Figure 2B), after which process the editing efficiency of 1.9-2.1x10^-6 was achieved, and a relatively effective transformation system of Chlamydomonas reinhardtii was established.


To verify whether the transfer of the vector into 503 cells of Chlamydomonas reinhardtii at the molecular level is successful or not, we used the colony PCR method to ensure full integration of the vector. We extracted the DNA from the above amplified algal solution after successful transformation by heating lysis at 95℃, and designed primers with a length of about 600bp from four mutually owned fragments of vector, namely Cas9 protein, Hyg resistance gene, mCherry reporter gene and StayGold reporter gene. The Hyg resistance gene and Cas9 protein were shared by the two vectors, and the reporter genes mCherry and StayGold were owned by vectors pTX2038 and pTX2040, respectively. For the PCR amplification reaction on the DNA of the transformed single algal colony, we also set up a linear plasmid of the vector as a positive control group.


The monoalgal colonies of pTX2038 and pTX2040 have been transferred. The brightness of PCR products of Hyg resistance gene, mCherry reporter gene, Cas9 protein and StayGold reporter gene (Figure 3) fragments were all consistent with the DNA bands of the positive control group as well as being matched with the position of DNA Marker, which indicates that the fragmented PCR products transferred into the vector were in a high concentration and normal expression state. In sum, the effectiveness of transformation could be testified.

To further demonstrate the correct insertion of the vector at the cellular expression level, we performed microscopy whose centers were the autofluorescent genes contained in the construct vectors: mCherry and StayGold. And autofluorescence in red of Chlorophyll was used as a negative control. It is worth noting that although both chlorophyll autofluorescence and mCherry will appear red after fluorescence excitation at certain wavelengths, they do not affect the observation because the wavelength range of their excitation of fluorescence does not overlap.


Compared with the wildtype, the cells of Chlamydomonas reinhardtii with fluorescent reporter gene showed different degrees of fluorescence and normal autofluorescence expression of chlorophyll, which further proved that the vector could be expressed normally when transferred into Chlamydomonas reinhardtii (Figure 4).

No synthetic biology research can be carried out without targets. Only when we identify the specific genes to edit, the newly constructed CRISPR system can show its power.

The goal of Chlipid is to increase TAGs accumulation in algae through synthetic biology to produce large amounts of biofuel. There has been a general consensus that algal TAG synthesis occurs primarily in response to stress such as nutrient deprivation; the most common of which is nitrogen limitation. The environment lacking nutrients or essential mineral factors such as stress would cause algal cells to undergo a series of physiological reactions, which involve the expression of a series of genes. Therefore, we suggest transcriptome sequencing and analysis would be a useful approach to locating target genes for further research and engineering.

Based on the design, we cultivated Chlamydomonas reinhadtii in nitrogen and iron stress conditions for 8h and 24h and then performed transcriptome sequencing and analysis to prove the idea. Throughout the process, we summarized the differential expression of four pathways and selected 12 genes with high/low expression (Table 1), which suggests using transcriptome sequencing and analysis can provide target sites for gene editing of lipid production in Chlamydomonas reinhardtii and the resources and information of genes for basic research and application research. ( More information on https://2022.igem.wiki/uestc-biotech/results)

Though we have successfully constructed a CRISPR system and used transcriptome analysis to locate target genes, generating CRISPR system to fulfil genome editing still needs a crucial step, selecting suitable gRNAs.

During the editing process, whether the Cas9 protein binds to an expected gene site for cutting is of great concern. A suitable guide RNA (gRNA) can efficiently reduce the likelihood of the off-target ability. To gain a deeper understanding of the physical mechanisms behind gene editing, we develop a thermodynamics-based model to predict the potential off-target activity of the selected guide RNA. And the model we use overcomes the above binary limitations of the previous physics-based binary off-target predicting model and considers off-targets effects with both high possibility and low possibility. ( More information on https://2022.igem.wiki/uestc-biotech/model)

1. Wase, N., Black, P. & DiRusso, C. Innovations in improving lipid production: Algal chemical genetics. Progress in lipid research 71, 101-123 (2018).
2. Greiner, A. et al. Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. The Plant cell 29, 2498-2518 (2017).
3. Eslami-Mossallam, B. et al. A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity. Nature Communications 13, 1367 (2022).