Novel CRISPR-based technology for rapidly screening genes to understand

Zebrafish—small, fast-growing organisms that share the same genes as humans—are important to many biologists, finding them uniquely effective for learning a variety of questions, from how organisms evolve, How the nervous system drives habits. Now, with a new expertise developed by the College of Utah scientists known as mic-drops, fish may be more effective for large-scale genetic research.

The MIC-DROP, which was developed under the leadership of chemical biologist Randall Peterson, PhD, dean of the You Well Being Faculty of Pharmacy, allows researchers to quickly consider a whole host of features to effectively implement the CRISPR gene enhancer system in zebrafish. Allows for deployment. bunch of genes in a single experiment.

The progress marks the first time that screens using robust, Nobel-prize winning CRISPR expertise have potential in any animal model. Already, Peterson’s crew has used MIC-drop to determine several genes that are critical to overall development and performance of guts. Their strategy and findings are reported on August 19, 2021, in the journal Science.

The CRISPR system is a programmable technology for modifying DNA. To use it, researchers introduce a DNA-cutting enzyme (often an enzyme known as Cas9) into cells, along with an RNA piece of information that tells the enzyme where to cut. This may be the first step in modifying the sequence of a gene, or simply turning off the gene.

The strategy has made gene enhancement faster, cheaper, and more accurate in zebrafish and other laboratory organisms — although, says Peterson, it has been difficult to examine too many genes at once.

To deactivate a gene in a zebrafish embryo, the researchers put together an information RNA that focused on that gene, then combined it with the Cas9 enzyme, loaded the answer into a needle, and detected the answer in the embryo. Injected rigidly calibrated quantity. In the event that they need to inactivate a particular gene in a particular embryo, they have to load an entirely new needle with a new Cas9/information RNA response.

MIC-Drop, which stands for Multiplexed Intermixed CRISPR Droplets, solves that problem by packing the elements of the CRISPR system into microscopic oil-capped droplets that can form collectively without mixing their contents. To arrange the display of multiple genes with MIC-Drop, researchers start by creating a library of information RNAs. Each information RNA is packaged in its individual droplet with the Cas9 enzyme. To maintain surveillance of the target gene, each droplet also contains a DNA barcode to trace its contents.

The team fine-tuned the droplets’ chemistry to ensure they remained safe and isolated, so droplets designed to focus on individual genes would be fused together and into the same needle. will be loaded. Under the microscope, the MIC-drop consumer injects one droplet into a zebrafish embryo, then attacks the following embryo and injects the following droplet.

The method would be repeated on multiple occasions, delivering a packet of CRISPR elements to each embryo, so that in each embryo, the system could inactivate a single gene. Then it is as much as if researchers have to look at animals for possible consequences.

Previously, it would have taken a team of researchers several days to establish a CRISPR display of the entire set of genes in zebrafish and needles, says postdoctoral researcher Saba Parvez, PhD, who developed and adapted the packaging of the MIC-drop. The whole group will be needed. Approach and barcoding system. “Now you can streamline that course into one consumer doing it in the span of a few hours,” he says.

To reveal the potential of MIC-Drop, Parvez and colleagues worked with U of Wellbeing collaborator H. Joseph Yost, Ph.D., Callum McRae, MD, PhD, Harvard Medical Faculty and Jing-Rue Joanna Ye. did. to investigate 188 completely different zebrafish genes for a possible condition in coronary heart enlargement at Massachusetts Basic Hospital.

After creating RNA that focused on these genes and introducing the CRISPR system into a whole group of fish embryos, they identified several animals that developed coronary heart defects as they matured. Using the DNA barcodes in these fish, the team was able to re-sign defects to 13 completely different inactivated genes. Because of the similarity between zebrafish and human genes, the discovery could lead to previously unknown features of coronary heart enlargement in people.

Peterson and Parvez are desperate to get the mic-drop to work in different labs, and so they say the 188-gen display is only a start. “Ultimately, individuals would really like to have the ability to do genome-scale screening,” Peterson says. “I believe that with this expertise the scale becomes really perceptible.

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