CRISPR: What's All the Excitement About?

A New Tool for Gene Manipulation

DNA sample being pipetted into multi well plate with DNA gel below
••• Rafe Swan/Cultura/Getty Images

Recently scientists have found an exciting new tool with which to engineer DNA. The CRISPR system has nothing to do with keeping your vegetables fresh in the refrigerator. It is the acronym for the newest system to manipulate genomic DNA in almost any animal. Researchers have been able to knock out or eliminate genes, repress gene expression, and up-regulate genes to increase expression with the CRISPR technology. It is a very flexible technique that researchers can use to easily alter the expression of genes to better understand their function.

What Exactly Is CRISPR?

CRISPR stands for Clustered Regularly-Interspaced Short Palindromic Repeats—an incredibly boring name for an exciting technology. Why the tedious name? It is because, when they were first discovered in the late 1980s in bacteria, no one knew what the short stretches of repeated DNA separated by random DNA sequences were for. They were just some strange feature in the genomic DNA of some bacteria.

It took almost 20 years until Jennifer Doudna at the University of California figured out that these sequences matched parts of certain viral DNA that infected the bacteria. As it turned out, the CRISPR sequences were a sort of immune system for the bacteria.

How Does It Work?

Doudna and her collaborator, Emmanuelle Charpentier, eventually worked out that, when infected by a virus, bacteria that had these short repeating DNA pieces that matched the viral DNA would use them to make RNA that bound to the DNA of the invading virus. Then, a second piece of RNA made from the random DNA that separated the CRISPR repeats interacted with a protein called Cas9.  This protein would cleave the virus DNA and inactivate the virus.

Researchers quickly realized they could exploit this capability of CRISPR to cut apart specific DNA sequences to knock out genes. While there are other techniques, such as zinc finger nucleases and TALENS that can be used to target and cut specific locations in genomic DNA, these approaches rely on bulky proteins to target the alternations to specific regions in the DNA. It is difficult to design and carry out modification on a large scale with lots of genes using these earlier approaches.

What Makes It so Useful?

The CRISPR system only relies on two short pieces of RNA: one that matches the targeted DNA region, and a second that binds to a protein called Cas9. In fact, though, it turns out that both these short RNA pieces can be combined into a dual-function single-guide RNA molecule that both targets a specific DNA sequence and recruits the Cas9 cleaving protein. This means that the Cas9 protein and one short piece of RNA that is 85 bases long is all that's needed to cut a DNA at almost anywhere in the genome. It is relatively simple to introduce DNA to produce a single-guide RNA and the Cas9 protein almost any cells making CRISPR generally applicable. 

However, convenient targeting is not the only advantage of the CRISPR technology over other TALENS and zinc fingers. The CRISPR system is also much more efficient than these alternative approaches. For example, a group at Harvard found that CRISPR deleted a targeted gene in 51%–79% of the cases, whereas TALENS efficiency was less than 34%. Due to this high efficiency, another group was able to use CRISPR technology to directly knock out genes in embryonic mice to produce transgenic mice in a single generation. The standard approach requires a couple generations of breeding to get the mutation in both copies of a targeted gene.

What Else Can It Do?

In addition to deleting a gene, some groups have also realized that, with a few alternations, the system can be used for other sorts of genetic manipulation. For example, at the beginning of 2013, a group from MIT showed that CRISPR could be used to insert new genes into genomic DNA. Shortly after that a group at UCSF used a modified version of the system dubbed CRISPRi to repress expression of target genes in bacteria. More recently, a group at Duke University also set up a variation of the system to activate sets of genes. Several groups are also now working with variations of these approaches to screen large numbers of genes at once to figure out which one is involved in different biological responses.

The Shiny New Toy of Genetic Engineering

Certainly, there is tremendous excitement about this new tool for genetic engineering and the rush to apply it for a variety of applications. However, there are still some challenges that need to be overcome and, as is often the case with new technology, it takes a while to work out where the limitations are. Researchers at Harvard, for example, have found that CRISPR targeting may not be as precise as initially thought. Off-target effects of the CRISPR complex can lead to unintended changes when altering DNA.

Despite the challenges, though, CRISPR clearly has shown enormous potential to facilitate alteration of genomic DNA that will help researchers more quickly understand how the tens of thousands of genes in the human genome function. This alone has important implications for improvements disease treatment and diagnosis. Further, with additional development, the technology itself may be useful for a novel type of therapeutics. It may provide a new approach for gene therapy. However, these advances are a ways off. For now, it is just exciting to watch the rapid development of this new research tool and think about the types of experiments it may allow.

(Posted: Sept 30, 2013)