Using CRISPR Technology for Allergy and Asthma
A new collaboration with Integrated DNA Technologies (IDT) to use their CRISPR-Cas9 gene-editing technology and the Alt-R® Human Kinase Library may enable discovery of new druggable targets for allergy.
The Sean N. Parker Center for Allergy and Asthma Research at Stanford University is very excited to collaborate with Integrated DNA Technologies (IDT) to use their CRISPR-Cas9 gene-editing technology and the Alt-R® Human Kinase Library. “We look forward to this highly impactful collaboration with IDT. We are excited to use their gift to enable discovery of new druggable targets for allergy,” said Dr. Kari Nadeau, Director of the Center.
CRISPR gene editing technology is an extremely powerful technology that has revolutionized biological research and enabled gene editing to become commonplace in the scientific world. It has enormous implications to human health. With this technology, one can easily cut DNA with unprecedented precision to create gene knockouts, alter genes, and screen the genome for genes that cause disease. Although CRISPR has mainly been used in research to understand diseases using cells and animal models, this technology is now being tested in human clinical trials in patients with cancer in China and in the USA. Dr. Nadeau is looking forward to this collaboration. “IDT is excited to assist Dr. Nadeau in bringing CRISPR screening technology to search for new therapeutic targets in the field of allergy and asthma. Methods already exist to perform high-throughput screens in primary human T-cells, making it an ideal time to embark on this project. I am confident that important new findings will emerge over the next few years,” said Dr. Mark Behlke, Chief Scientific Officer of IDT.
The CRISPR-Cas9 system has 2 main components – a search and identification tool (guide RNAs, denoted as gRNAs) and a cutting or snipping tool (Cas9 endonuclease). gRNAs are small pieces of genetic material that can be easily pre-designed to bind a specific gene of interest within a cell. By identifying and binding the gene of interest, gRNAs direct Cas9 to the precise region of DNA that needs to be deleted or edited. When Cas9 cleaves the gene specified by the gRNA, repair mechanisms come into action to fix the newly formed break in the DNA. One method includes end-joining the 2 strands together - an error-prone method that causes genes to become dysfunctional allowing scientists to create gene knockouts. Disrupting a gene or creating knockouts can be a very powerful tool in understanding the function of specific genes.
In allergy, T helper 2 cells (Th2) cells are the immune cells known to increase in number and become activated to secrete inflammatory molecules (cytokines). These cytokines then activate another immune cell type (B cells) to secrete IgE, a key substance involved in allergic reactions. When IgE binds to mast cell, a person is said to be sensitized to that allergen. When a sensitized person is exposed to allergens, mast cells immediately begin to secrete a number of molecules, such as histamine, that mediate symptoms of allergy.
In our lab, using our repository of tissue samples obtained from our twin cohort and other mechanistic studies, Senior Researcher Dr. Swati Acharya along with other collaborators have identified key genes associated with Th2 cells that are likely involved in allergic disease. By creating gene knockouts with CRISPR, we hope to determine the precise role of these genes in allergic disease. Th2 gene knockout cells will be assessed for cell expansion, as well as levels of secretion of key inflammatory biomarkers (IL-4, IL-5, IL-9, IL-13). Data obtained from these knockout studies along with data obtained from other cutting–edge technologies, such as ATAC-seq, RNA-seq, and CyTOF, which together give us information as to which genes are accessible for being translated into protein and which proteins are being expressed, can provide us a more complete picture of allergic pathways.
CRISPR cleavage results in a double-stranded break in cellular DNA that must be repaired. Standard biochemical end-joining that reconnects the 2 cut strands is error-prone and often leads to gene knockouts. Another method of fixing the damage is by inserting a specific short strand of DNA at the spliced region. Scientists, by tailoring and supplying specific DNA templates to the cell, can alter a gene as desired or correct a mutation. This cutting edge technology has far-reaching implications. Once we have confirmed the role of key genes involved in allergic disease with gene knockouts, we hope to use this knowledge to edit these genes and reprogram cells to prevent allergic reactions.
IDT offers several CRISPR libraries that can further our knowledge of novel genes associated with allergic disease through the use of high throughput screening of the genome. For this first project, IDT provided the Center with a library of 3400 gRNAs that target more than 800 human kinase genes. While any subset of the genome is limited by definition, kinases are a major drug target and a major control point in cell behavior and can provide a useful starting point. As of November 2016, the FDA has approved 31 kinase inhibitors for human use for treatment of various diseases. During CRISPR screening, target cells are treated with constructs from a CRISPR library to create a population of mutant cells that are then evaluated for specific changes in cell expression or structure. Drs. Nadeau and Acharya are excited to collaborate with IDT and use this technology to further evaluate the samples obtained from our twin cohort to determine differences in key regulatory T cell genes in those with and without allergies and asthma.
By Vanitha Sampath
Vanitha Sampath received her PhD in Nutrition from the University of California at Davis. At the Sean N. Parker Center for Allergy and Asthma Research, as a medical writer and content manager, she enjoys being in the midst of groundbreaking research in asthma and allergy and is committed to communicating the scientific advances of the Center and spreading awareness of its mission and vision.