CRISPR-based technology to accelerate the identification of genes involved in health and disease

August 19, 2021 12:00

Zebrafish, small, fast-growing creatures that share many of the same genes as humans, are essential to many biologists, who find them uniquely suited to studying a wide range of questions, such as how organisms develop the way the nervous system determines behavior. Now, thanks to a new technology developed by scientists at the University of Utah called MIC-Drop, fish will be even more powerful for large-scale genetic studies.

MIC-Drop, whose development was led by chemical biologist Randall Peterson, Ph.D., dean of the faculty of pharmacy at U of U Health, enables researchers to efficiently deploy the CRISPR gene editing system in zebrafish to quickly assess the functions of hundreds of genes in a single experiment. This breakthrough marks the first time that displays using robust, Nobel-winning CRISPR technology are possible in any animal model. Already, the Peterson team has used MIC-Drop to identify several genes essential for healthy development and functioning of the heart. Their method and results are reported on August 19, 2021 in the journal Science.

The CRISPR system is a programmable method of DNA modification. To use it, researchers introduce a DNA-cutting enzyme (usually an enzyme called Cas9) into cells, along with an RNA guide that tells the enzyme where to cut. This can be the first step in changing the gene sequence, or just shutting down the gene.

The method has made gene editing in zebrafish and other laboratory organisms faster, cheaper, and more accurate – but, says Peterson, it has been difficult to scale up to study more than a few genes. at a time. To inactivate a single gene in a zebrafish embryo, researchers prepare a guide RNA targeting that gene, then mix it with the Cas9 enzyme, load the solution into a needle, and inject a carefully calibrated volume of the solution into the embryo. . If they want to inactivate a different gene in a different embryo, they must load a new needle with a new solution of Cas9 / guide RNA. “The process has always been focused on one gene or one modification at a time,” says Peterson. “So if you want to do 100 genes, that’s 100 times more work.”

MIC-Drop, which stands for Multiplexed Intermixed CRISPR Droplets, solves this problem by wrapping CRISPR system components in microscopic oil-coated droplets, which can mix without mixing their contents. To set up a screening of many genes with MIC-Drop, the researchers start by creating a library of guide RNAs. Each guide RNA is packaged in its own droplet, with the Cas9 enzyme. To keep track of target genes, each droplet also includes a DNA barcode identifying its content.

The team refined the chemistry of the droplets to ensure they remain stable and unobtrusive, so that droplets designed to target different genes can be mixed and loaded into the same needle. Under a microscope, the MIC-Drop user injects a single droplet into a zebrafish embryo, then moves on to the next embryo and injects the next droplet. The process can be repeated hundreds of times, delivering a single package of CRISPR components to each embryo, so that in each embryo the system inactivates a single gene. Then it’s up to the researchers to monitor the animals for potential effects.

Previously, setting up a CRISPR screen of hundreds of genes in zebrafish would have required a team of researchers several days and hundreds of needles, says postdoctoral researcher Saba Parvez, Ph.D., who developed and optimized the packaging technique and the barcode of MIC-Drop. system. “Now you’ve streamlined this process to a single user who does it in a matter of hours,” he says.

To demonstrate the potential of MIC-Drop, Parvez and colleagues worked with U of U Health colleague H. Joseph Yost, Ph.D., Calum MacRae, MD, Ph.D., at Harvard Medical School , and Jing-Ruey Joanna Yeh, Ph .D., at Massachusetts General Hospital to test 188 different zebrafish genes for a potential role in cardiac development. After creating guide RNAs targeting these genes and introducing the CRISPR system into hundreds of fish embryos, they identified several animals that developed heart defects as they matured. Using DNA barcodes from these fish, the team was able to trace defects to up to 13 different inactivated genes. Due to the similarities between zebrafish and human genes, the discovery may point to previously unknown aspects of heart development in humans.

Peterson and Parvez are eager to see MIC-Drop implemented in other labs, and they say screening for 188 genes is just the start. “At the end of the day, people would like to be able to do genome-wide screening,” says Peterson. “I think this scale becomes really imaginable with this technology.”