CRISPR is an incredibly useful tool for geneticists and microbiologists. Short for clustered regularly interspaced short palindromic repeats, it sounds intimidating on the basis of name alone. Yet it’s a tool that Annie Taylor—a senior applied and engineering physics major at Cornell University—uses extensively in her research. What she does with CRISPR showcases some of its powers.
“I can write out a nucleotide sequence on my computer, buy a custom-made DNA fragment for that sequence, and use it to program the CRISPR system to target practically any DNA sequence I want with high specificity.” She explains. Hearing how she uses it, it’s easy to imagine that CRISPR has had a tremendous impact in biological research, with applications in countless subdisciplines.
Creating Genetic Circuits to Operate Like Computer Circuits
Taylor conducts her research in Guillaume Lambert’s lab, Applied and Engineering Physics, which focuses on creating genetic circuits in cells—an application of CRISPR in synthetic biology. Genetic circuits are analogous to logic circuits in electrical engineering. Logic circuits are comprised of many logic gates; they alter the voltage and current of a system as it passes through them. Similarly to how computers function, genetic circuits may eventually be used to perform complex logic functions in a cell.
Many types of logic gates perform a variety of functions. Taylor is working on a particular type of logic gate known as a NOT gate, or an inverter. The input of the gate is at a high voltage, while the output of the gate is at a low voltage (or vice versa). In electrical engineering, high voltage is represented by one, while low voltage is zero. A one at the input of a NOT gate would produce a zero at the output. The same is true the other way around.
In biological systems, there is no high voltage or low voltage. Instead, one and zero would correspond to high and low protein expression. A one would mean that a certain protein is expressed, while a zero would mean the protein is inactive.
“While the systems and the techniques are biological, a lot of the underlying theories of our work revolve around physics.”
In a cell, protein expression can be easily examined. One can insert marker genes such as the green fluorescent protein (GFP) gene. A cell that is expressing GFP will glow green in ultraviolet light or blue light, so one can tell whether or not GFP is being expressed by simply shining a blue light over the colonies. Taylor inserts these marker genes into a cell by inserting them into a plasmid, a structure that carries genetic information, which is then absorbed and incorporated into bacterial cells. The end result is a colony of cells that glows green in blue light. Taylor then uses the CRISPR system to target this inserted GFP gene.
How CRISPR Works
“The CRISPR system, in nature, is a bacterial immune system. Bacteria are trying to protect themselves from viruses, which are basically sequences of foreign DNA. They have to be able to recognize what genetic information is foreign and what genetic information is their own.”
To do this, bacterial cells produce Cas proteins, which bind to a guide RNA sequence. Then the Cas protein inspects any DNA it encounters. If any sequences on the DNA match with the guide RNA sequence, the Cas protein recognizes it as being foreign DNA and destroys it.
The most commonly used Cas protein for gene editing, Cas9, natively acts to cut DNA at the place where it is identical to the guide RNA sequence, but Cas9 can be modified in many ways. For instance, the Lambert lab often uses dead Cas9—known as dCas9)—which has been modified so that it is catalytically inactive. When it recognizes a DNA sequence, instead of cutting the DNA, dCas9 binds to that sequence and inhibits transcription. The target DNA sequence is not cleaved but becomes unable to express the protein for which it codes.
The power of the CRISPR system lies in the fact that one can modify the 20-base pair guide RNA sequence that the Cas9 protein binds to. If one knows a critical sequence of DNA on a gene of interest, they can create a corresponding guide RNA sequence that will cause the Cas9 protein to target that area of DNA. If it is dCas9 protein, then it will bind to that sequence of DNA and inhibit transcription, the expression of a protein at that site.
Taylor can design a guide RNA sequence that she knows will correspond to a sequence on one of the marker genes she inserts. For example, she can create a sequence of RNA that corresponds to a critical part of the GFP gene. Before she inserts the guide RNA sequence, the bacterial cells will express the GFP gene. Afterward when the dCas9 has bound to the RNA sequence, recognized the corresponding sequence on the DNA, and deactivated it, the colonies will no longer produce GFP and will no longer glow under ultraviolet light.
“There are a variety of Cas proteins,” Taylor explains. “Each Cas protein may have a different guide RNA structures or may deactivate genes in different ways. And while dead Cas9 inhibits transcriptions, there are other ways to modify Cas proteins to activate genes. It gives us control over which genes are expressed in a cell and which are inhibited.”
Creating Genetics Circuits
One of the aims of the Lambert lab is to use this control over gene expression in order to generate a genetic circuit. They want to develop a system in which they can submit certain inputs, and knowing how the logic gates in the cell will interact with the inputs, be able to obtain a predictable result.
Living systems are incredibly complex, however. Having multiple logic gates might cause them to interfere with each other’s function. The Lambert lab is working to understand the limitations of the system. How many logic gates can be inserted until their behavior is no longer predictable and stable? What are the off-target effects of the CRISPR system?
“If you have a genome that is millions or billions of base pairs long, there might be several locations that the Cas protein will unintendedly target, because they match the 20-base pair guide RNA sequence simply by chance,” Taylor says. “This might be disastrous for the cell, or the cell might appear to be unaffected. It just depends on where those off-target sequences are and what they code for.”
The Fusion of Physics and Biology
Taylor appreciates the intersection between sciences, which is the focus of the Lambert lab. She entered Cornell interested in aerospace engineering, yet in her senior year she has found a passion for combining physics and biology. Minoring in biological sciences, Taylor has been able to apply her coursework to her lab work.
“While the systems and the techniques are biological, a lot of the underlying theories of our work revolve around physics,” she says. She hopes to use the gene editing and manipulation tools that she has learned through her work with the Lambert lab in her future research.
CRISPR is both a scary name and a powerful tool, and as Taylor has learned through her work with the Lambert lab, its applications to scientific research are widespread. It has the potential to turn genetic loci into logic gates and cells into circuits, to turn proteins on or off at will, and to inform our growing knowledge of how cells—and life—function.