Introduction to CRISPR Genome Editing

What Is CRISPR?

CRISPR (pronounced "crisper") is the acronym for Clustered Regularly Interspaced Short Repeats, a group of DNA sequences found in bacteria that act as a defense system against viruses that could infect a bacterium. CRISPRs are a genetic code that is broken up by "spacers" of sequences from viruses that have attacked a bacterium. If the bacteria encounter the virus again, a CRISPR acts as a sort of memory bank, making it easier to defend the cell.

Discovery of CRISPR

The discovery of clustered DNA repeats occurred independently in the 1980s and 1990s by researchers in Japan, the Netherlands, and Spain. The acronym CRISPR was proposed by Francisco Mojica and Ruud Jansen in 2001 to reduce the confusion caused by the use of different acronyms by different research teams in scientific literature. Mojica hypothesized that CRISPRs were a form of bacterial acquired immunity. In 2007, a team led by Philippe Horvath experimentally verified this. It wasn't long before scientists found a way to manipulate and use CRISPRs in the lab. In 2013, the Zhang lab became the first to publish a method of engineering CRISPRs for use in mouse and humane genome editing.

How CRISPR Works

Essentially, naturally-occurring CRISPR gives a cell seek-and-destroy capability. In bacteria, CRISPR works by transcribing spacer sequences that identify the target virus DNA. One of the enzymes produced by the cell (e.g., Cas9) then binds to the target DNA and cuts it, turning off the target gene and disabling the virus.

In the laboratory, Cas9 or another enzyme cuts DNA, while CRISPR tells it where to snip. Rather than use viral signatures, researchers customize CRISPR spacers to seek genes of interest. Scientists have modified Cas9 and other proteins, such as Cpf1, so that they can either cut or else activate a gene. Turning a gene off and on makes it easier for scientists to study the function of a gene. Cutting a DNA sequence makes it easy to replace it with a different sequence.

Why Use CRISPR?

CRISPR is not the first gene editing tool in the molecular biologist's toolbox. Other techniques for gene editing include zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and engineered meganucleases from mobile genetic elements. CRISPR is a versatile technique because it's cost-effective, allows for a huge selection of targets, and can target locations inaccessible to certain other techniques. But, the main reason it's a big deal is that it's incredibly simple to design and use. All that's needed is a 20 nucleotide target site, which can be made by constructing a guide. The mechanism and techniques are so easy to understand and use they are becoming standard in undergraduate biology curriculums.

Uses of CRISPR

Researchers use CRISPR to make cell and animal models to identify genes that cause disease, develop gene therapies, and engineer organisms to have desirable traits.

Current research projects include:

• Applying CRISPR to prevent and treat HIV, cancer, sickle-cell disease, Alzheimer's, muscular dystrophy, and Lyme disease. Theoretically, any disease with a genetic component may be treated with gene therapy.
• Developing new drugs to treat blindness and heart disease. CRISPR/Cas9 has been used to remove a mutation that causes retinitis pigmentosa.
• Extending the shelf life of perishable foods, increase the resistance of crops to pests and diseases, and increase nutritional value and yield. For example, a Rutgers University team has used the technique to make grapes resistant to downy mildew.
• Transplanting pig organs (xenotransplanation) into humans without rejection
• Bringing back woolly mammoths and perhaps dinosaurs and other extinct species
• Making mosquitoes resistant to the Plasmodium falciparum parasite that causes malaria

Obviously, CRISPR and other genome-editing techniques are controversial. In January 2017, the US FDA proposed guidelines to cover the use of these technologies. Other governments are also working on regulations to balance benefits and risks.

Selected References and Further Reading

• Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P (March 2007). "CRISPR provides acquired resistance against viruses in prokaryotes". Science. 315 (5819): 1709–12. 
• Horvath P, Barrangou R (January 2010). "CRISPR/Cas, the immune system of bacteria and archaea". Science. 327 (5962): 167–70.
• Zhang F, Wen Y, Guo X (2014). "CRISPR/Cas9 for genome editing: progress, implications and challenges". Human Molecular Genetics. 23(R1): R40–6.