Engineering, as commonly suspected, is to design and build certain products to meet certain needs. Engineering a biological system is not much different. First, we shall identify our needs. The question of What and Why should we build hit us way before the project started. We tried to locate a significant topic that can be solved by building a biological system. This year, after witnessing the deterioration of the world situation, unusual climates appear much more frequently, and continuous fights for resources in many areas, we chose to focus on energy issues and related climate issues.

As we have introduced in the project description, we plan to design a biological system, either based on biological traits already present or create one from scratch, that can produce biofuels using microalgae as chassis. Microalgae accumulate TAGs, an important storage lipid, to maintain energy homeostasis and carbon storage. Attempts to use genome engineering to increase TAGs accumulation have been done for years, however, there hasn't been much success due to the complexity of algal metabolic pathways.

Though the aim seems hard to achieve, we are soon devoted to designing our own biological system. To start with, we need to decide on the specific microalgae as the chassis. We consulted our PIs, who proposed we target Chlamydomonas reinhardtii, the classic chassis among microalgae since it's the first time for us and the PIs to engineer microalgae. However, Chlamydomonas reinhardtii has multiple strains. After comparing data from the Chlamydomonas Resource Center of the University of Minnesota and the Freshwater Algae Culture Collection at the Institute of Hydrobiology of China, we chose CC-125 (the basic “137c” wild type strain) and CC-503 (a very clean wall-deficient mutant) as candidate strains. For more than a month, we cultured both strains in standard TAP media and drew growth curves describing the relationship between OD750 (reflects cell number) and time. To better analyze algae cultivation efficiency, we introduced the following formula by conducting a series of experiments and stimulations to construct the correspondence between cell number and OD value.

We found that CC-503 has a larger population size and a longer lifespan than CC-125, not to mention that CC-503 is wall-deficient which make it easier to be transformed. We then decided Chlamydomonas reinhardtii strain CC-503 as the final chassis.

Chosen the chassis, the next step is to select genetic methods and engineer a matching system. Based on a review named Innovations in improving lipid production : Algal chemical genetics by Nishikant Wase, we noticed that many attempts were done with overexpression of positive regulation genes in TAGs or fatty acids synthesis pathways but few have tried to knock down or knock out positive regulation genes in glucose synthesis pathways or TAGs degradation pathways. Combined with our careful investigation and previous experience of the team, we supposed this is a good year for us to dive again into CRISPR system to see what can we achieve. Among the large Cas protein family, we selected the S. pyogenes (Sp) Cas9 endonuclease as our gene-editing tool.

The moment we chose to use CRISPR/Ca9 system, we knew there was a problem that can not be neglected, off-target. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively and rely on binary classification schemes to identify strong off-targets. Therefore, we had to construct a model that could provide a quantitative assessment of the off-target effects of different sgRNAs to guide and offer better choices on gRNAs so that we could know precisely which gRNAs to synthesize that could make the most out of our transformation process and guarantee gene editing efficiency. Our modelling group made a marvellous contribution to this matter.

They constructed a kinetic model based on thermodynamics which can give more discriminative off-target scores for better differentiation of different gRNAs compared to MIT and CFD scores. And the model we used can overcome the binary limitations of the previous physics-based binary off-target predicting model and considers off-target effects with both high possibility and low possibility. And an idea of accurately calculating the cleavage efficiency of a certain sgRNA based on the Moran process is given by us.

Although we did suspect building a knock-out system targeting algae will be difficult, the available results were still surprisingly insufficient. We selected a CRISPR/Cas9 plasmid designed to target photoreceptor genes in Chlamydomonas reinhardtii, according to Andre Greiner, as the foundation to engineer our own parts. The origin design of the plasmid seems to perfectly fit our needs but we want to insure all the parts are capable to fulfil our aim of knocking out different genes located in algal nuclear genome in vivo. Therefore, we performed codon optimization, kept 2 different promoters for gRNAs, and replaced reporter genes with mCherry and Staygold (a newly discovered highly photostable green fluorescent protein). pTX2038 and pTX2040 are designed.

We chose the commonly used electronic transformation to transform pTX2038 and pTX2040 without cloning specific gRNAs but remaining gRNAs inserting site into algae.

The brightness of PCR products of Hyg resistance gene, mCherry reporter gene, Cas9 protein and StayGold reporter gene (Figure 3A and 3B) 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.

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 (Figure4).

We learned from this first attempt that transforming CRISPR plasmids into algae is not a smooth process. Our team tried a protocol that describes a method of transforming Chlamydomonas reinhardtii nuclear genome through electroporation. This scheme requires at least 3 days of work, which usually leads to small colonies within 1-2 weeks, but no single colony is successfully screened after many attempts. We struggled for months to get the result of the plasmids transformation. We began to question our design on the methods.

In order to improve the efficiency of electric conversion, we increased the amount of linear plasmid, improved the parameters of electroporation, and used electroporation buffer (ME Suc): MAX efficiency ™ Convert the reagent (Therfisher, # A24229) to make further attempts.

For the plasmids, we noticed that although we have successfully proved that Cas9 protein and the reporters were transformed into algae, we only had concrete test results on reporters. Cas9 protein hasn't had the chance to show its ability! We found that psy1 (phytoene synthase-1 gene, disruption of psy1 produces white colonies that are easy to detect and count) is a valuable and mind-blowing reporter gene which is an ideal in vivo target gene for us to attempt our first transformation towards Chlamydomonas reinhardtii.

We used the tool called CRISPOR on the sgRNA design website, together with the off-target prediction model we constructed, to perform sgRNA design for psy1. The following are the test results on psy1 knock-out.

Comparison between wildtype Chlamydomonas reinharditii and transformed positive clone of PSY1 gene in bright field(Figure 5).
Here, we provide a scheme to verify the successful introduction and proper operation of the CRISPR/Cas system by targeting the endogenous PSY1 gene for vector construction and transformation. Moreover, by microscopic bright-field comparison of the wild type with the PSY1 mutant, if the field of view turns white, the system could prove to be feasible.

According to previous research, scientists have found that algae accumulate more TAGs when they are cultured in deprived conditions such as nutrition deficiency, temperature or light treatment and many researchers have conducted transcriptome analysis towards those deprived treated algae. The most use one is nitrogen deprivation, which play an important role in regulating gene expression and metabolic pathway. Iron is also an essential micronutrient for all organisms and plays an important role in the growth and metabolism of Chlamydomonas reinhardtii. However, research on iron stress in Chlamydomonas reinhardtii is relatively blank. Therefore, we chose conventional stress - nitrogen stress and novel stress - iron stress among various stresses to observe their effects on the growth and lipid production of Chlamydomonas reinhardtii. And we managed to identify several differential expressed genes (Table 1).

Now we managed to find some target genes by our own approach but we couldn't neglect the fruits of previous generations so we browsed more than 50 relative papers to observe more important enzymes in algal metabolic pathways that may have huge impacts on TAPs accumulation. From previous research, we selected 7 genes from 5 different pathways as possible target genes and from our transcriptome analysis results we predicted 12 genes as possible targets (Figure 6). We shall knock out each of them using our engineering system and then test these mutants' growth conditions and TAGs accumulations.

We planned to continue drawing growth curves as done before, and use Nile red dying, lipid extraction and GC-MS to quantitively and qualitatively analyze TAG and fatty acids accumulation so that we could create engineered algae to produce a large amount of biofuel raw material in a fragmentation tank like a factory.

1. 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).
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. Wase, N., Black, P. & DiRusso, C. Innovations in improving lipid production: Algal chemical genetics. Progress in lipid research 71, 101-123 (2018).