Careful Cuts with CRISPR Turn Genes On or Off

At Michigan State University, Cheryl Rockwell, assistant professor of pharmacology and toxicology, studies the immune cells that defend us from foreign materials. In particular, Rockwell explores how our immune system responds to environmental dangers, such as arsenic and cadmium. These contaminants activate a protein—nuclear factor erythroid 2-related factor 2 (Nrf2)—that sets off a process of detoxification, protecting us from the contaminants. On the other hand, Nrf2 drives the creation of tumors in some cases. To determine exactly what drives Nrf2 to help or hurt us, Rockwell needed a way to control the gene that makes it. To do that, she turned to CRISPR.

Part of a bacteria’s defense against foreign DNA, such as that injected by a virus, depends on having clustered regularly interspaced short palindromic repeats (CRISPR) in its genome. The CRISPR DNA gets transcribed into CRISPR (guide) RNA that combines with CRISPR-associated (Cas) proteins to turn off the foreign DNA by cutting it into an unusable form. Scientists now take advantage of targeted cutting by CRISPR/Cas systems by using it to turn off specific genes.

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To study genes most closely, scientists need tools that activate or inhibit the pathways. “In our lab,” says Rockwell, “we have used CRISPR/Cas9 to silence the expression of a number of genes.” One of those is the gene for Nrf2. By making a model system in which Nrf2 can be controlled, Rockwell and her colleagues hope to unravel Nrf2’s helpful effects from the detrimental ones.

On or off

Scientists can pick from CRISPR activation or inhibition—CRISPRa and CRISPRi, respectively—to turn a gene on or off. “The main applications for CRISPRa and CRISPRi are gene expression studies,” says Michael Castelli, product manager for PCR, cell technology and detection at Eppendorf. “To understand the function of a gene or to reproduce the conditions of certain diseases, it becomes necessary to turn gene expression on—overexpression; or off—knockdown or knockout.” In brief, CRISPRi blocks gene transcription and CRISPRa activates or enhances it.

Before the advent of CRISPR, scientists used plasmids to transfect a cell with several copies of a gene, to overexpress it. That enabled the cell to produce more protein encoded by that gene. “By contrast, CRISPRa allows for expression of the gene using the endogenous genomic sequence, which presumably allows for ‘natural’ processing of the mRNA and expression of downstream isoforms of the gene,” says Joel McDade, senior scientist at Addgene.

To turn down gene expression, scientists used a technique called RNA interference (RNAi). Now, they can also use CRISPR to remove the gene or inactivate it. “Methods to study a reduction in expression of a given gene have been limited to short-hairpin RNA and short-interfering RNA approaches,” McDade says. “One of the unique aspects of CRISPRi is that one is able to turn down expression of a given gene without completely destroying the genetic code that encodes it.” He adds, “This is also advantageous when trying to study genes for which complete loss of the gene is toxic.”

Delivery devices

To put CRISPR to work, scientists need the right tools. That starts with reagents. “Addgene now carries CRISPRa/i reagents that can be used to turn genes on and off in a wide variety of species, including mammals, bacteria, yeast, plants and fly,” McDade says.

There are many kinds of Cas proteins, and different ones can be used with CRISPR.

In some applications, one kind of Cas is more effective than another, and researchers can now purchase reagents that use various types of Cas proteins.

Scientists also need tools to put the genetic changes in the right place. Eppendorf, for instance, makes micromanipulation and microinjection instruments that scientists can use to target individual cells for CRISPR-mediated modifications. “These techniques allow you to manipulate the expression levels of a gene by modifying transcription at the endogenous sequence,” Castelli says. “This is an important distinction when compared to techniques that would utilize exogenous sequences to generate the same result.”

In addition, the CRISPR approach is “far less expensive and more available,” Castelli says.

Analyzing efficiency

Although CRISPR makes it easier than ever to edit a genome, it remains imperfect.

One team of scientists reported that “quantification of resulting gene knockout rates still remains a bottleneck,” and they developed a method using digital PCR for fast and accurate quantification [1].

According to Carolyn Reifsnyder, senior marketing manager of the digital biology group at Bio-Rad Laboratories, “Droplet digital PCR, ddPCR, is precise, low-cost and has a fast turnaround time.” With ddPCR, the reaction takes place in a droplet. In fact, one sample gets divided into 20,000 droplets, which increases the precision and sensitivity of this technique over other forms of PCR.

With ddPCR, says Reifsnyder, scientists can reveal editing events below 1% of a cell line; real-time PCR only gets down to about 5%. “Most people using CRISPR do some sort of validation,” Reifsnyder explains. Some scientists, for example, sequence the genome after CRISPR editing, but ddPCR can be used, too. “We have half a dozen groups validating CRISPR edits with our ddPCR solution, which includes a brand new custom assay design portal,” Reifsnyder says.

To simplify the use of ddPCR to validate CRISPR edits, Bio-Rad developed a website where a scientist enters the sequence of interest and the specific organism used, and the company sends the necessary ddPCR assay. With this, scientists can test a sample before moving forward with other experiments. “If you carry a cell line forward that was not edited or was edited at the wrong site, you waste valuable time and money,” Reifsnyder says.

Being able to edit genes more precisely than ever—particularly to turn them up or down—provides scientists with an array of new ways to explore life. We can see how systems behave when healthy, and how they fail. We can watch systems under attack and, perhaps, learn to defend or repair them. As of March 30, 2017, a search of ClinicalTrials.gov found CRISPR used in seven studies, but that number is set to skyrocket.

Reference

[1] Mock, U, et al., “Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases,” Nature Protocols, 11:598-615, 2016 [PMID: 26914317]