Drosophila
Drosophila, often called fruit flies, samples under a microscope.
Credit: Tyanna Lovato

One of education’s biggest challenges is keeping up with technology to ensure that students are familiar with the latest technological advances and enabling them to be successful during their academic careers and beyond. This is especially true in the physical sciences where technology goes hand in hand with research.

One such technical advance involves new genome editing technology used to manipulate genome sequences and gene expression. CRISPR, or clustered regularly interspaced short palindromic repeats, is a natural defense mechanism found in a variety of bacteria. Scientists have adapted CRISPR into a new technology that has come online in the last half dozen years or so, enabling scientists to efficiently edit genomes with flexibility and precision.

It’s a technique that allows scientists to manipulate a DNA sequence in a very specific and controlled way. The process involves a CRISPR RNA (crRNA) and a CRISPR associated gene (Cas9),  that induce double-stranded DNA breaks at a targeted location that can lead to gene mutation in the DNA, or controlled alteration of the DNA sequence.

Traditionally, researchers might have used chemicals or radiation to make random changes in a DNA sequence. Then, they’d have to go in and figure out what changes were made, which is time intensive. With CRISPR, it’s possible to make changes to very specific locations which is an advantage because scientists can target individual genes they’re working on and or interested in, in order to understand more about how the genes work in the life of the animal.

Mutation Prep
Students are involved in each step of the process including injecting the DNA to create the mutations.

Students in Professor Richard Cripps’ Biology class 498L/598L are using the new technology to rapidly create gene mutations by generating the short crRNA molecules that in turn target specific DNA sequences through complementary based pairing. The CRISPR technology, which continues to emerge, has been adapted for genome editing in a variety of systems including the fruit fly Drosophila, which Cripps utilizes in his class.

“For general research purposes this is an amazing innovation because for researchers like us, it enables us to find a gene we’re interested in and study its function,” said Cripps. “We can knockout that gene and see what the effect is and then it tells us the function of the gene. We can do that a lot more quickly and directly than we’ve ever been able to do before.

“On a biomedical front it allows us the potential to target mutations causing cancer or repair mutations that cause muscular dystrophy and other genetic diseases. CRISPR has an unprecedented potential in medicine and cancer biology and in realms of finding ways to cure inherent diseases.”

"I know of no other place doing this, so kudos for kick-starting the program at UNM.”  – Dr. Joseph Miano, professor, Department of Medicine, University of Rochester Medical Center

Given the ability and the speed in which genes can be mutated, Cripps, who introduced the class initially in Spring 2015, decided to put the technology to the test in a single-semester in an undergraduate class. The goal was to have students isolate mutants for specific genes in fruit flies using the CRISPR technology.

“In the laboratory we’re assigning students a gene of interest, and they get to design a target sequence within that gene and knock it down,” said Dr. Tyanna Lovato, a research scientist working with Cripps. “They get to perform a variety of tasks including computer work, molecular techniques and Drosophila genetics to get hands on experience in knocking down a gene. By the end of the semester they should be able to determine whether the gene knockout worked or not and characterize the mutation if it did.”

Students are very involved in the process including injecting the DNA to create the mutations. “They get a lot of hands on experience both in the laboratory and in class through lectures that teaches them about the technology and mechanisms behind it,” added Lovato.

Preparing microscope slides
A student prepares flies for viewing under a microscope..

In the class, six students were each assigned a single Drosophila gene where no mutants existed. Each student designed and created plasmids to encode crRNAs targeting their selecting gene. The plasmids were then injected into Cas9-expressing embryos that enable students to delete the selected gene. Students then conducted a two-generation cross to test for heritable transmission of a mutated allele and generated a stable stock of mutant alleles characterized by PCR and sequencing. Three out of six genes ended up successfully mutated.

Chauncey Gadek, a senior majoring in biology and chemistry minor, attempted to delete a Drosophila  named TroponinC, responsible for flight muscle function. Gadek said it’s a fairly straightforward process that begins with the design of nucleotide targets for the crRNA Cas9 complex to cut flanking either side of the target gene. It ended with nine lines of flies that exhibited the expected phenotype for a deletion of the TpnC4 gene (they were unable to fly).

“Though sequencing revealed I had not excised the target gene, the CRISPR system had caused two small, yet powerful loss of function insertion/deletions,” Gadek said. “The mutation was originally observed in flight tests of the fly crosses.”

Carol Deaton, a fifth-year senior majoring both in biochemistry and biology, was impressed with the CRISPR technology even though she also was unable to mutate a gene. 

“I was not able to mutate a gene because my oligonucleotide constructs did not cut the DNA efficiently,” Deaton said.  “We were not able to determine this until I sequenced groups of flies.”

Even though Deaton was unsuccessful, the class and the techniques she learned will be quite valuable including significant research experience in order to merit offers from graduate programs.

Tyanna Lopez and Richard Cripps
Research Scientist Tyanna Lovato (l.) and Professor Richard Cripps have been called "visionaries" for the introduction of CRISPR genome editing technology into an laboratory setting at UNM.

“I am very impressed with the biological construct of the CRISPR technology and the story of its discovery and impact is amazing,” Deaton said. “It has introduced me to techniques I will use to earn my Ph.D.” Deaton said. “The technology itself is a powerful tool in my research. I may not only be able to improve evaluation of genetic diseases, but find ways to cure them. As a future physician scientist, the lessons I learned in this class, including critical thinking and problem solving, are crucial.”

Since the CRISPR technology is very new, this is one of the first classes in the world where undergraduates are taught the technique hands-on. The class is already garnering national interest, based upon publication of its planning and execution in the journal Biochemistry and Molecular Biology Education.

Dr. Joseph Miano, a professor in the Department of Medicine at the University of Rochester Medical Center, is impressed with the development of the class. “I know of no other place doing this, so kudos for kick-starting the program at UNM,” Miano said. “Moreover, the millennial generation will be growing up with CRISPR technology as a viable medical option, so it is important that they are well informed and experienced with the technology.”

Lovato and Cripps are teaching the class for a second time this spring, training a total of 16 students in the cutting edge technology. They plan to also offer it in future semesters. “There are still plenty of Drosophila genes to mutate,” said Lovato, “and in the future we might expand the training to use other model organisms.”

In this way, UNM instructors are ensuring that its graduating students remain at the forefront of their fields.