Photo credit: Aaron Blanco Tejedor
Tell me this: What does the friendly neighborhood Spider-Man and my research have in common?
Genome editing.
Genome editing makes specific changes to the blueprint of life, otherwise known as DNA, of a cell or an organism. If you’ve kept up with the late Stan Lee’s Spider-Man comics or the Spider-Man movies, you know that Peter Parker was bitten by a radioactive spider that changed his DNA, resulting in him having amazing spider-like abilities. As much as I’d like to believe that a radioactive spider bite can alter a human’s genome, giving him or her amazing spider-like abilities, as a scientist I must tell you that it is highly unlikely (sorry to the dreams out there I just crushed).
However, genome editing is possible.
This past year I joined the lab of Dr. Richard Cripps at San Diego State University, where I first encountered the genome editing tool called CRISPR; a topic you may have heard of in the news. In the lab, we use CRISPR-Cas9 technology (Cas9 is the acronym for CRISPR-associated protein 9) to mutate specific genes and examine how these mutant genes affect the normal progression of muscle development and muscle function in the fruit fly Drosophila melanogaster.
Photo credit: Philip Veater
By using fruit flies, we can study many generations, over a few weeks, at a low cost. In addition, there are parallels between human and fruit fly genomes. Some genes associated with human genetic disease are found in the fruit fly genomes. By targeting those genes, scientists are able to study the role of those genes with the hope of finding better treatment(s) to prevent or cure human disease.
Specifically, we seek to advance our knowledge about the role of transcription factors in muscle development. Transcription factors are proteins that bind to a specific DNA sequence and either turn the gene “on” or “off”. They make sure that the genes are turned “on” or “off” at the right time and in the right place. Otherwise, chaos ensues, much like when Thanos collected all six infinity stones. Okay … maybe not to that extent, but some serious issues can arise from transcription factor failure such as skeletal muscle disorders.
Many of the skeletal muscle disorders that affect people worldwide (e.g. muscular dystrophies and inflammatory myopathies) arise from genetic mutations. To date, treatment exists to alleviate some of the symptoms (e.g. muscle weakness, movement issues, and twitching) associated with these disorders, however, some treatments (e.g. immunosuppressant medicines) pose a higher risk to some individuals by weakening the body’s immune system, so people with compromised immune systems are placed at a higher risk.
In my lab, we hope that our findings using CRISPR as a tool will be able to improve treatment strategies to slow or halt the progression of skeletal muscle disorders.
Photo credit: David Boca
How does CRISPR work?
To understand how CRISPR works, we first have to understand how DNA gives rise to everything that makes up you. You may be thinking, what makes me … me? It’s genes! Genes make up who you are. Genes are made up of DNA, passed on from parent to offspring, carrying specific instructions that determine the offspring’s traits, such as eye color and hair color, among other things.
CRISPR is the acronym for Clustered Regularly Interspaced Short Palindromic Repeats.
The CRISPR system contains a few parts, the first of which is called a single guide RNA (sgRNA), the second of which is called Cas9. You can think of the sgRNA as similar to Google Maps. Let’s say you input the address for In-N-Out, a mouth-watering burger joint. The “address” you input for the sgRNA to take you to is the DNA target sequence that you are interested in, or where you want to make your mutation. When Google Maps (the sgRNA) has arrived at its location, it signals to the Cas9, which can cut the DNA like a molecular scissors (Figure 1).
Figure 1. Recognition of the PAM (short Protospacer Adjacent Motif) by the Cas9 nuclease. Credit: IDT
Once Cas9 recognizes the target DNA sequence and it matches with the sgRNA, the Cas9 will cut both DNA strands, 3-4 nucleotides before the PAM site (Figure 1). Think of PAM as the In-N-Out attendant that directs you towards the In-N-Out entrance. In this case, PAM ensures that Cas9 makes the cut at the correct location, slightly upstream of the targeted DNA site (Figure 1).
After both strands of DNA are cut, the cell’s innate ability to repair itself will kick in. One of the cell’s repair mechanism is to bind the broken double-strands of DNA back together, but often, errors arise along the way. These errors may result in additional nucleotide(s) or a deletion of nucleotide(s) at the DNA target site (Figure 2). These errors may also lead to a frameshift mutation which shifts the way the DNA sequence is read, ultimately, altering the amino acid chain and affecting protein structure and function (Figure 2). However, this actually works to our advantage, because the goal of this experiment it to disrupt the gene of interest so that the transcription factor (aka protein) that is produced does not function properly; remember, we WANT a mutant!
Figure 2. Non-homologous end joining (NHEJ) resulting in nucleotide insertion, deletion, or frameshift mutation. Credit: Medium.com
Because the transcription factor (aka protein) functions improperly in our newly engineered mutant fruit flies, these genetically edited mutants may or may not have the ability to fly, jump, or move normally. We can then compare the mutant flies to wild-type (or un-mutated) flies that have normally functioning genes, and look for differences between the two fly lines. These differences will give us an insight as to how specific genetic mutations affect the resulting protein’s function and how this change affects the normal progression of muscle development and function. Later, we can apply this to humans.
Photo credit: Aryan Singh
A day in my CRISPR life.
To generate the CRISPR-Cas9 system in our fruit flies, we inject plasmids that encode for a target specific sgRNA, into one-hour old transgenic Cas9 fly embryos.
A plasmid is a circular DNA strand in the cytoplasm of a bacterium that can make many copies of itself. Thus, making many copies of our target specific sgRNA and ebony sgRNA.
First, we collect our transgenic embryos from grape juice agar plates in fly cages (see below, left). Secondly, we remove the “eggshell” with 50% bleach, and align the embryos in the same orientation on a fresh grape juice plate (see below, center). Thirdly, we transfer the embryos from the grape juice agar plate to a desiccator for 6.5 minutes, and cover them with oil. Lastly, we microinject the embryo’s posterior end with the plasmid containing the target specific sgRNA and ebony sgRNA in injection buffer (see below, right). The ebony sgRNA will help us screen for ebony (dark body colored) fruit flies from the surviving microinjected flies. These ebony flies most likely carry the mutation.
Fruit fly cages and a grape juice agar plate with yeast paste (left). Fly embryo aligned and ready to be transferred onto a microscope slide (center). Fly embryo micro-injections (right).
Significance and versatility of CRISPR
Note, that without Cas9, the CRISPR-Cas9 system is incomplete and we would be unable to mutate the gene of interest. If the microinjections are successful, the gene of interest will be mutated, and mutant flies will arise bearing an ebony phenotype.
Fruit flies are great model organisms because they have a short life span, high reproduction rates, and a simpler genome compared to vertebrates. In our lab, we are able to generate targeted mutations and successfully engineer a fruit fly’s genome within three months.
How can CRISPR benefit us?
Presenting my research poster at the 2019 Future Faces of STEM alongside the SDSU SACNAS (Society for the Advancement of Chicanx/Hispanics and Native Americans in Science) officers.
The current industries that use CRISPR-Cas9 technology include healthcare, agriculture, and transportation. CRISPR brings hope to people worldwide because it could be a great tool for preventing or correcting genetic errors that cause muscle disorders as well as other human diseases. In agriculture, CRISPR is used to increase crop production and even generate reduced-gluten wheat for those who are gluten intolerant. Scientists are even using CRISPR to manipulate the genome of bacteria and algae to produce biofuels. CRISPR could help resolve the world’s energy crisis.
CRISPR technology is growing fast, and will continue to do so because it is less expensive and more accurate than previous DNA editing techniques. However, with a technology this powerful, it is important to understand and control off-targets (unintended genetic modifications), which must happen before successfully using CRISPR-Cas9 technology in humans. For now, we hope that using this technology in fruit flies will take flight!
-Elizabeth
Photo credit: Road Trip with Raj