In Chlipid, we tend to construct a biological system that uses CRISPR/Cas9 system to manipulate Chlamydomonas reinhardtii metabolic pathways to increase algal inner TAGs accumulation so that to gain raw materials for biofuel production. To get a better idea of how Chlipid should and would be implemented in the real world, we researched the current state of the industry, identified application prospects and anchored application targets through multiple routes. Combining what we have learned from the real world and what we have achieved in the lab, we propose the following implementation methods.

We suggest the following 2 types of end users for our project.

In Chlipid, we primarily focus on developing a profound genetic editing system based on CRISPR/Cas9 technology for detailed research on algae lipid metabolism, which is extremely complex. We also provide the idea of performing transcriptome analysis linked to stress cultivation as a powerful approach to discovering, selecting and determining target genes. Through the system, researchers can obtain a huge number of mutants all with the potential to accumulate more TAGs than wild-type algae and grow normally. These mutants are not only precious experimental materials but also possible commercialized products. Mutants carrying differently edited genes may also produce TAGs with diverse fatty acids composition and content, for example, TAGs that are rich in palmitic, oleic, and linoleic acids guarantee biofuels with better quality, which it's another key point while conducting biofuel research.

Using microalgae to produce biofuel is still a developing yet immature industry. Its outstanding advantage of biofuel production and greenhouse gas absorption catches the attention of investors. However, current relative mature technologies in the field still need excessive costs which prevent large-scale industrialization (more information on communication). Chlipid can serve as a rising star that solves the problem. Biofuel companies can be inspired by the research thoughts of Chlipid, engineer their own super algae, or use the mutants we create to produce biofuel raw materials.

As introduced in the project description, the boundary between implementing genetic engineering in microalgae to increase the accumulation of TAGs and real-world industrialization is the high costs, therefore, breakthroughs in core production technologies are the only hope for real-world implementation. The core technological challenge is that TAG biosynthesis in algae, like higher plants, is a complex process, more than 100 genes have been identified to be involved in lipid metabolism not to mention some enzymes are multifunctional and carry out two or more activities which differ as algae living environment shifts. Also, TAG biosynthesis in algae coincident with growth, which occurs primarily in response to stress such as nutrient deprivation coupled with a slowing of cell growth and reproduction that have to be overcome to make algal biofuels commercially viable.

From previous research, we believe that under stressful living conditions most commonly nitrogen deprivation, algal carbon flux shifts into storage molecules such as starch and TAGs. Also, we took essential micronutrients such as iron into consideration to see whether micronutrients affect algal carbon flux key elements. We cultivated Chlamydomonas reinhardtii CC-503 within nitrogen and iron deprivation respectively. Then took algae samples after cultured for 8h and 24h, the samples were then sent for transcriptome analysis to identify differential expressing genes in diverse metabolic pathways under stress conditions.

Such a method is an indispensable component of the foundational research system that acts as a guide for end users to understand and locate their tension in the massive metabolic network they seek to use.

Another important part of the foundational research system is the genome editing tool we use. We manage to build a genome editing system based on the CRISPR-spCas9 system that can not only achieve single but also multiple knock-out actions. The system is specially designed to target genes in Chlamydomonas reinhardtii nucleus genome. End users can use the CRISPR off-target model we constructed or other gRNAs design tools to design gRNAs. In the system, we use hygromycin resistance protein and 2 optional fluorescent proteins as reporters which provides more diversity while implementation. With the system, end users can manipulate target genes we have found but have not yet completed measurement or other genes that they are interested in.

Based on the foundational research system, we are able to target genes located in lipid metabolism individually and combinatory, therefore, a mutant bank is built. Then, we monitor each mutant's growth by drawing growth curves for qualitative evaluation. To quantitively and qualitatively measure TAG accumulation in algae, we perform Nile red dying, weighing method, TAG and fatty acids separation and extraction, and GC-MS to analyze fatty acids components. Mutants that not only grow normally but also produce the highest amount and best quality TAG for biofuel production are potentially engineered super algae suitable for large-scale bio-fermentation.

Biofuel is derived from the TAGs that algae accumulate. The triacylglycerols are converted to fuel by trans-esterification with methanol. The suitability of these fuels for particular climatic conditions depends largely on the mix of fatty acids in the triacylglycerols. Transesterification of TAGs is also a necessary process for researchers to analyze fatty acids composition and content of TAGs extracted from algae using GC-MS or LC-MS. Chlipid is built to produce more biofuel as renewable energy. Undoubtedly, biofuel processing and production are one of our proposed implementations not to mention that the entire production process is theoretically free of greenhouse gas emissions and even absorbs greenhouse gases already in the atmosphere, perfectly avoiding the climate crisis that could result from energy mining and use.

In addition to absorbing greenhouse gases, microalgae have other valuable environmental functions such as generating electricity, releasing oxygen, treating sewage, degrading plastic, made into biodegradable materials for clothes, furniture or straws. Chlipid's foundational research system, a genetic editing tool based on CRIPSR/Cas9, makes it easier to add more desired functions to the project.

Take sewage treatment as an example. Research on wastewater treatment by means of microalgal-bacterial processes has become a hot topic worldwide during the last two decades. Owing to the lower energy demand for oxygenation, the enhanced nutrient removal and the potential for resource recovery, microalgal-based technologies are nowadays considered a good alternative to conventional activated sludge treatments in many instances. Such technology inspired us to use sewage as an alternative culture media for algae. All we need to do is to perform sewage component analysis and sterilization, then sewage can be tested and used as a cultivation approach. With the method, Chlipid has another function of treating sewage.

The natural benefits of microalgae can be added to existing projects in a variety of ways.

Though Chlipid guarantees multiple proposed implementations, there are several challenges that remained.

We have successfully proved that single knockouts can be done using Chlipid foundational research system (more information on proof of concept), however, the multi-knockout plasmid still needs to be designed. What's more, we haven't done enough measurements over the designed process of mutant research, including culturing in fermentation tanks, drawing growth curves, lipid extraction for TAGs isolation and fatty acids composition analysis, and biofuel in lab production so that before putting Chlipid into real-world implementation, there's still more to be added.

Using CRISPR/Cas9 system as the genome editing tool makes many security issues need to be taken into account. One of which is to contain Cas protein inside algae to prevent possible leakage that may accidentally cause unintended editing of random organisms. We have designed a kill switch with team Sorbonne_U_Paris to achieve the goal (more information on safety). Microalgae have high tolerance and adaptability to the environment, making them easy to grow everywhere with basically water and sunlight, therefore, addressing the importance of safety also prevents environmental issues like water bloom from happening.

1. Bryant, J. A. Fatty Acids, Triacylglycerols and Biodiesel. Biofuels and Bioenergy, 105–118 (2017).
2. Quijano G, Arcila JS, Buitrón G. Microalgal-bacterial aggregates: Applications and perspectives for wastewater treatment. Biotechnol Adv. Nov 1;35(6):772-781(2017).