CRISPR/Cas9-based gene knockouts allow for a better understanding of lipid metabolic pathways.
About the authors
Olga (Olya) Vvedenskaya, Kateryna Ivanova
Olga (Olya) Vvedenskaya
Sci. Communications Officer
Dr. Dr. Olya Vvedenskaya studied medicine, and further obtained her PhD in the field of molecular oncology. She loves to deliver scientific messages in a clear and accessible manner.
Kateryna Ivanova
Research Associate (IFW Dresden)
Kateryna holds a master's in Molecular Bioengineering. She is eager to see and comprehend how biology is influencing many facets of our lives, from fashion to medical.
• CRISPR/Cas9-based gene knockout is used to investigate enzyme activities and the molecular lipid composition
• Cell growth density influences the lipid composition
• Functions of certain lipids can be taken over by other lipids
Olga (Olya) Vvedenskaya
Sci. Communications Officer
Dr. Dr. Olya Vvedenskaya studied medicine, and further obtained her PhD in the field of molecular oncology. She loves to deliver scientific messages in a clear and accessible manner.
Kateryna Ivanova
Research Associate (IFW Dresden)
Kateryna holds a master's in Molecular Bioengineering. She is eager to see and comprehend how biology is influencing many facets of our lives, from fashion to medical.
THE omics sciences play a crucial role for the analysis of gene expression, proteins, metabolites, and lipids variations. However, the investigation of how different metabolic pathways are regulated in health and disease is challenging. While the process to identify the genomic bases of diseases and connect them to protein and enzyme malfunctions is well established, lipidomics still lacks specificity in defining which enzymes and proteins are responsible for generating and regulating different lipid species.
CRISPR-Cas9 is a gene-editing technology that allows scientists to cut and modify DNA sequences with high precision and efficiency, making it a powerful tool for genetic research. Briefly, CRISPR-Cas9 works by using a guide RNA to target a specific DNA sequence, which is then cut by the Cas9 enzyme, allowing for precise genetic modifications.
Permanent loss-of-function cell lines were generated by CRISPR/Cas9 gene trap and deletion methods, resulting in independent knockout lines for 23 lipid-related genes. Afterwards the knockout cell lines grown to a confluency of 80% were selected for a more detailed lipidome analysis, since an initial principal component analysis (PCA) of 2095 lipid species within 25 lipid classes has shown that more reproducible and reliable data were obtained at this confluency, compared to 20% confluence.
Principal component analysis used for data analysis.A A score plot generated by a principal component analysis (PCA) on all lipids found in at least one cell line and density replicate (n=1809), with the colors indicating the cell density (20% or 80% confluence) in the petri dish. B The PCA score plot utilizes all lipids found in at least one cell line replicate (n=1449), with only samples generated using the gene trap method at 80% confluence being used and colored by the gene targeted. Highlighted and named genes separated from the bulk and control samples. The percentage of variability explained in each principal component is indicated in the axis labels. C The PCA score plot utilizes all lipids found in at least one cell line replicate (n lipids=1590), with only samples generated using the deletion method. The percentage of variability explained in each principal component is indicated in the axis labels.
Spiegel et al., Scientific reports (2022) 12:10533, 10.1038/s41531-022-00335-6.
Firstly, the authors analysed the effects of single-gene knockout cell lines within the sphingolipid class by targeting ceramide synthase 2 (CERS2) which is mainly responsible for the synthesis of ceramides with C20-C26 fatty acids. As expected, the knockout led to a reduction in sphingolipid species with such fatty acid lengths. A subsequent increase in sphingolipids with C16 fatty acids is indicative of a compensation due to the introduced changes. Moreover, additional changes impacted a third of all lipid species, significantly differentiating CERS2 knockout cell lines form the control in the PCA. This highlights the importance of complete lipidome analysis to assess lipid metabolism changes that result from the loss of a single protein.
Lipid profiles of CERS2 knockout cells.Left CERS2 knockout lipid profile changes represented in a volcano plot. Lipid species of the CERS2 knockout cell line and the control were compared. Outlined points indicate lipids significant after correction for multiple testing. Right the distribution of sphingolipid length in CERS2 knockout cells. Lipid species have been normalized to the lipid class and summed up by their total number of carbon atoms (shown on the x-axis). Standard deviations are visualized with error bars. * for q<0.05, ** for q<0.01,**** for q<0.0001. Cer, ceramide; HexCer, hexosylceramide; SM, sphingomyelin.
Spiegel et al., Scientific reports (2022) 12:10533, 10.1038/s41531-022-00335-6
It was also observed that different knockouts might lead to similar effects. This is the case of sphingomyelin synthase (SGMS1), which catalyses the reaction yielding sphingomyelin and diacylglycerol (DAG). This knockout has led to a reduction of total SM level, which has led to the upregulation of a compensatory pathway, since the total hexosylceramide (HexCer) increases. A similar phenotype is observed with Mannose-P-dolichol utilization defect protein (MPDU1), where the HexCer increases and SM decreases.
Also, multiple gene knockout of fatty acid metabolism was tested. Knockouts of two genes, ELOVL5 and HSD17B12, which have sequential activity in the long-chain fatty acid elongation, were analyzed.
Fatty acid elongation pathway. Four enzymatic steps ensure the sequential elongation of long-chain fatty acids, allowing for the synthesis of diverse fatty acid species in living organisms. The extension of the fatty acid (n) by two carbon atoms (n+2) is finalized with the fourth step.
ELOV1-7, Elongation of very-long-chain fatty acids; KAR, 3-ketoacyl-CoA reductase; HADC, 3-hydroxyacyl-CoA dehydratase; TER, trans-2,3-enoyl-CoA reductase.
Spiegel et al., Scientific reports (2022) 12:10533, 10.1038/s41531-022-00335-6
The knockout cell lines overall show a similar fatty acid profile within lipid classes, confirmed by a high degree of correlation of the ratio of lipid classes compared to control. In particular, the similarity of phosphatidylcholines (PC) and phosphatidylethanolamine (PE) and their plasmalogens, ether-linked phospholipids, profiles was observed.
Selected lipid and fatty acid profiles of HSD17B12 and ELOVL5 knockouts. Individual profiles of PC, PE and their plasmalogens with fatty acid profiles. Error bars visualize to standard deviations. Adjusted p-values visualize error bars. * for q<0.05, ** for q<0.01, *** for q<0.01, **** for q<0.0001. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PC O-, ether-linked phosphatidylcholine; PE O-, ether-linked phosphatidylethanolamine.
Spiegel et al., Scientific reports (2022) 12:10533, 10.1038/s41531-022-00335-6
This proof-of-concept study demonstrates that quantitative lipidomics screens can produce reliable and reproducible data and that these screens should be further expanded to more genes and different cell lines. As a goal, it will be possible to create a comprehensive map of lipid pathways at the molecular level including proteins involved in each step of the lipid metabolism. This information is of crucial importance for developing new diagnostic and therapeutic approaches.
The common research principle at the MPI-CBG is to dissect fundamental cellular functions and to identify their generalizability and uniqueness in development and evolution that result in diverse and distinct tissues and organisms. Their research programs span the scales from molecules to organisms and continuously evolves new angles that reflect a vibrant and evolving landscape.
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