“Charpentier and Doudna's technology has revolutionised life sciences. In just eight years, it has evolved from an interesting discovery to a tool which is used as a standard in laboratories around the world – also at the Department of Biology and Biological Engineering. Nobel Prizes are usually awarded to discoveries that are older as they needed more time to gain ground, says Elin Esbjörner, Associate Professor of Chemical Biology, whose research group studies protein aggregation mechanisms and amyloid formation in neurodegenerative diseases.
Briefly, this is how gene editing with CRISPR-Cas9 works: The enzyme Cas9 is the scissors that cut DNA. For the scissors to cut at the correct position in the genome, a piece of RNA is constructed, a single-stranded molecule that matches the sequence on the DNA molecule to be cut.
This RNA sequence guides Cas9 to the target with high precision. The cut of the DNA strand makes it possible for the researchers to turn off certain genes – and thus certain functions in the cells – or to paste new genes into the genome, which provides the cells with new properties.
ALS research and studies on nucleic-acid based drugs
“We use the genetic scissors to label cells, to light up the proteins we want to study. We achieve this by adding a gene sequence that extends the protein with a so-called fluorescent, light-emitting, marker. In this way, we can study the protein directly in living cells using a microscopy. This is a very powerful way to understand biology and how the body works. We use our genetically modified cell models to investigate a protein that causes the neurodegenerative paralysis disease ALS (amyotrophic lateral sclerosis), but also to study how nucleic-acid based drugs are taken up and distributed inside cells,” says Elin Esbjörner.
She says that the genetic scissors provide more opportunities to examine, in a finely tuned manner, why some people suffer from neurodegenerative diseases, such as Parkinson's and Alzheimer's.
“It enables us to work with models that are similar to the conditions of the disease. Previously, we had to modify our cell models with a method that involves expressing the proteins we study in very high concentrations. The high protein concentration enhances the risk that the results do not sufficiently represent what is really happening in the patient’s brain.”
New Wallenberg Academy Fellow seeks to prevent neurodegenerative disorders
43 Chalmers researchers receive funding for more research
Development of efficient cell factories
Florian David, Assistant Professor at the Division of Systems and Synthetic Biology, uses the gene scissors for a completely different area of research: the development of cell factories.
A cell factory is a microorganism, often yeast, that can be genetically adapted for large-scale and sustainable production of a variety of products, such as biofuels, drugs and chemicals. For these cell factories to be used industrially, they must be viable, productive and efficient, under the industrial conditions.
Florian David's research group develops new CRISPR based tools to efficiently engineer strains of baker’s yeast, Saccharomyces cerevisae, for sustainable production of chemicals and novel drugs.
The cell factories are genetically optimised to ferment renewable sources of sugars, which can for instance be derived from waste products from the wood industry. Through this fermentation process the cells can produce various products. Cell factories can be a key in the transition from an oil-based to a bio-based industry, but one major challenge is to make the production cost-effective.
“For me, efficiency and precision are the big advantages of CRISPR-Cas9. We do not want long development cycles because it is too expensive – consuming both time and money. This technology has accelerated development and optimisation of yeast strains considerably,” says Florian David.
He believes that from societal and environmental perspectives, it is important that the development of these cell factories is fast and efficient for the industry.
“If we succeed in making the different steps of the production cheaper, we will move more quickly, towards a change in the industry enabling sustainable production and environmentally friendly products based on renewable resources,” he says.
In one of his research projects the CRISPR-Cas9 technology was used to create thousands of different yeast strains in a short time frame and screen for the most efficient producers using high throughput screening approaches. More insights on how to improve the yeast cell factory are gained quickly, directly speeding up the development cycle.
Model-assisted fine-tuning of central carbon metabolism in yeast through dCas9-based regulation
Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape.
Development of cell factories and tools for new organisms
Yvonne Nygård, Associate Professor at the Division of Industrial Biotechnology, also develops cell factories that use residues from forestry and agriculture to produce biofuels and biochemicals. Her research group runs several projects that are based on CRISPR-Cas9, and others where the technology is used as one of many tools for genetic modification.
“Cutting and pasting genes was the first application, but the technology has developed continuously, and now there are many applications. You can now use the technology for up- and down-regulation of genes, which means that you can control the activity of different genetic pathways in the cells. We have just published a study (link below) where we have developed a so-called tool kit, a system, where a variant of the CRISPR-Cas9 technology is used to regulate genes in industrial yeast strains,” she says.
Yvonne Nygård's group also develops CRISPR-based tools for filamentous fungi, and her previous research involved the development of tools for the fungus that is used industrially to produce penicillin.
“The great thing about CRISPR-Cas9 is that the technology works in many different organisms. Before the genetic scissors, you had to develop the genetic toolbox from scratch if you wanted to work with a new organism, which can be time consuming. These days you can relatively easily implement the same technology and tools in different types of cells,” she says.
Yvonne Nygård explains that it is now much easier to test different production organisms in parallel - or to start using new microorganisms found in nature.
“The CRIPSR-Cas9 technology provides better conditions for working with organisms that are difficult to manipulate genetically, such as filamentous fungi. Filamentous fungi grow relatively slowly and have several cell nuclei, all of which contain chromosomes, which requires effective genetic modification tools,” she says.
A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12
CRISPR-Based Transcriptional Activation Tool for Silent Genes in Filamentous Fungi
Potential ethical issues when using CRISPR-Cas9
The researchers are of the same opinion regarding potential ethical issues of the CRISPR-Cas9 technology: It is not the method itself that involves ethical dilemmas, it depends on how it is used. For example, using the technique clinically, as treatment of various diseases, may involve many ethical issues.
“There are probably no direct ethical, CRISPR-Cas9-related problems associated with my or the other BIO researchers' studies, as the cells engineered with this technology do not differ from cells engineered with other tools. But it would be naive to say that there are no ethical issues connected to the technology. You never know what the tools you develop will be used for,” says Yvonne Nygård.
Elin Esbjörner agrees.
“The genetic scissors have great potential when it comes to correcting genes directly in the body. This is fantastic, because we will probably be able to use the technology to cure serious diseases that we have not yet managed to cure with traditional medicines. But there are complex ethical issues regarding which genetic defects should be corrected. As members of the scientific community, we have a great responsibility to ensure that this fantastic technology is used, and will be used, for the proper purposes,” she says.
Text: Susanne Nilsson Lindh
This is how we use CRISPR-Cas9:
Verena Siewers, Senior Researcher, Systems and Synthetic Biology:
“My research is about developing cell factories for industrial production of chemicals, pharmaceuticals and lipids. We mainly work in baker’s yeast, and in this organism the gene scissors are an established technique for gene engineering, and by now my research group uses it in all projects for engineering yeast.
For me, the true beauty of CRISPR/Cas9 is the wide range of possibilities to modify the original technique. You can for example target a modified Cas9-protein to a specific position in the DNA, and instead of cutting the DNA it can regulate the expression of that specific gene − or create random mutations to evolve a certain region of the DNA. We have used this for genes involved in the fatty acid metabolism, but also to create so called libraries where we randomly altered gene expression in the cells – and then screened for expression patterns that were beneficial for a specific product. There are still some limitations to the technique, but our research would definitely have progressed slower without it.”
Model-Assisted Fine-Tuning of Central Carbon Metabolism in Yeast through dCas9-Based Regulation
Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols
Cecilia Geijer, Assistant Professor, Industrial Biotechnology:
“In my research group we develop industrial yeast strains, which can effectively convert sugars from plant biomass into sustainable biofuels and biochemicals.
Baker’s yeast, Saccharomyces cerevisiae, is a very efficient producer of bioethanol from glucose. In my group we use the CRISPR-Cas9 technology to introduce genes from other organisms into the genome of baker’s yeast, which also enables fermentation of other sugars from plant biomass and broadens the yeasts' spectrum of applications.
The gene scissors speed up the development process of these yeast strains considerably - and have a lot of advantages compared to other methods. For example, it is a very precise method and we can now modify the strains without introducing markers such as antibiotic resistance genes in the genome, which is a great advantage for yeast strains that will be used industrially.
I also work with new, relatively unknown yeast species that possess many industrially attractive properties. I am convinced that CRIPSR-Cas9 will be an essential tool for turning these "non-conventional" yeasts into efficient cell factories in the future.”
New network improves European yeast research
Genomic and transcriptomic analysis of Candida intermedia reveals the genetic determinants for its xylose-converting capacity
Fredrik Westerlund, Professor, Chemical Biology:
“In my research group, we use the gene scissors in an antibiotic resistance project to analyse plasmids. Plasmids are the "extra" DNA molecules of bacterial cells where the genes that encode antibiotic resistance often are found. Traditional methods of studying plasmids do not provide all the information that is clinically important. By using CRISPR-Cas9 in combination with a mapping method that we have developed, we can locate antibiotic resistance genes on specific plasmids.
We use the CRISPR-Cas9 technology in its simplest form: we cut the DNA and with the mapping method we identify if the DNA was cut, and in that case where; a cut only occurs if the antibiotic resistance gene is present.
Since plasmid DNA is easily picked up by other bacteria, compared to chromosomal DNA, a resistance gene on a plasmid can be expected to spread. Although PCR can determine if a specific strain has a resistance gene, it may be necessary and relevant for further studies to know exactly where the gene is located. If so, our analysis method is an excellent choice.”
Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping
Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak
Oliver Konzock, PhD Student, Systems and Synthetic Biology:
“In my research project, I am optimising the non-conventional yeast Yarrowia lipolytica for sustainable production of food oils, such as the high-value product cocoa butter.
Yarrowia lipolytica is naturally producing a lot of fat, however, the exact composition is different from my target food oils. To change the lipid composition, I use the gene scissors to remove or exchange the gene for a protein that induces double bonds between carbons in the fat molecules. By changing the gene or its expression level I can alter the fat composition and get closer to food oils.
For me, CRISPR/Cas9 is an extremely important tool, as it speeds up the gene engineering part of the project and I can spend my time testing the strains − or build more.“
Deletion of MHY1 abolishes hyphae formation in Yarrowia lipolytica without negative effects on stress tolerance