Nandini Pharasi, Jaypee Institute of Information Technology
CRISPR/Cas9 is a method that allows for the extremely precise and fast alteration of DNA in a genome, which is an organism’s full set of genetic instructions. This illustration demonstrates genome editing. CRISPR is a programmed technique of altering DNA. To utilize it, researchers insert a DNA-cutting enzyme (typically Cas9) into cells, along with an RNA guide that directs the enzyme where to cut.
Zebrafish and Gene editing
Zebrafish are tiny, fast-growing creatures that have many of the same genes as humans. They are especially well suited for researching a wide variety of topics, from how organisms develop to how the nervous system governs behavior. The technique has made gene editing in zebrafish and other laboratory creatures faster, cheaper, and more accurate, but it has been challenging to scale up to investigate more than a few genes at a time. Peterson, an author of a recent study published in the journal Science, said: “As recorded in the past, scientific progress only focuses on one gene or one modification at a time, so if you want to have 100 genes, you have to work 100 times more.”
MIC-Drop and its use
The fish is predicted to be even more potent for large-scale genetic investigations because of a new technique created by University of Utah Health experts called MIC-Drop. MIC-Drop is an abbreviation for Multiplexed Intermixed CRISPR Droplets. MICDrop users inject one drop into the zebrafish embryo under the microscope, then move to the next embryo and inject the next drop. The procedure may be repeated hundreds of times, each time sending a single packet of CRISPR components to each embryo, allowing the system to inactivate a single gene in each embryo. The researchers will then be responsible for monitoring the animals for any potential consequences. Researchers can now easily deploy the CRISPR gene editing system into zebrafish to rapidly test the functionality of hundreds of genes in a single experiment using MIC-Drop. This method overcomes the problem by encasing the CRISPR system’s components in small oil-encased droplets, which can mingle without contaminating their contents.
This might be the first step in changing the sequence of the gene, or it could just turn the gene off. Researchers produce a guide RNA targeting that gene, mix it with the Cas9 enzyme, load the solution onto a needle, then inject the solution into the embryo, which will silence a single gene in a zebrafish embryo. They must load a fresh needle with a new Cas9/guide RNA solution if they want to inactivate a different gene in a different embryo.
Construction
To build up a MIC-Drop screen comprising multiple genes, researchers first create a library of guide RNAs. Each guide RNA, together with the Cas9 enzyme, is packed into its droplet. To keep track of target genes, each droplet has a DNA barcode that identifies its contents. The chemistry of the droplets was fine-tuned to ensure that they remained stable and distinct, allowing droplets designed to target various genes to be combined together and put into the same needle. According to Saba Parvez, a PhD researcher who invented and enhanced the MIC-packing Drop’s method and barcoding system, before putting up a CRISPR screen of hundreds of genes in zebrafish would have required a team of researchers several days and hundreds of needles. “You’ve now reduced that process to a single user who can do it in a few hours,” he says.
Demonstration of the potential of MIC-Drop
To show MIC-potential, Drop’s Parvez and colleagues tested 188 distinct zebrafish genes for potential involvement in heart development at Massachusetts General Hospital. They discovered many animals that acquired heart abnormalities as they matured after generating guide RNAs targeting those genes and injecting the CRISPR system into hundreds of fish embryos. The researchers were able to track the problems back to 13 separate inactivated genes using the DNA barcodes in those fish. Because of the similarities between zebrafish and human genes, the discovery might hint at previously undiscovered elements of human heart development.
Peterson and Parvez, co-authors of the study in discussion, were reported to be delighted to see that MICDrop was being used by other labs, and believed that screening for the 188 genes was just the beginning. Peterson said that in the future, people will be able to do genome sequencing on a large scale by screening. “I imagine that with this technology, scalability can now be attained.”
Also read: Straglr: A new and efficient DNA sequencing technology
References:
- Parvez, S., Herdman, C., Beerens, M., Chakraborti, K., Harmer, Z. P., Yeh, J.-R. J., MacRae, C. A., Yost, H. J., & Peterson, R. T. (2021). MIC-Drop: A platform for large-scale in vivo CRISPR screens. Science. https://doi.org/10.1126/science.abi8870
- Sciences, U. of U. H. (n.d.). New CRISPR-based technology to speed identification of genes involved in health and disease. Retrieved August 21, 2021, from https://phys.org/news/2021-08-crispr-based-technology-identification-genes-involved.html
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