The genome-editing technology CRISPR has been lauded as one of the most important scientific breakthroughs of this century. CRISPR allows us to precisely insert, add, or delete genes inside living cells. Although similar technologies have existed for years, CRISPR is emerging as the clear winner due to its extraordinary precision, efficiency, and flexibility. It is enhancing our ability to improve crop yields and accelerating biomedical research by improving disease models and drug discovery, and it is poised to revolutionize our ability to treat genetic diseases.

Here, we will provide a brief overview of how this technology works, a history of the field to date, and a summary of future applications. The video below, PreScouter’s CRISPR webinar, provides more information about CRISPR.

CRISPR Is Part of a Bacterial Immune System

The history of CRISPR dates back to 1987, when Japanese scientists came across unusual repeating sequences in the E. coli genome. In subsequent years, researchers discovered similar repeats in other species of bacteria and archaea. Because these sequences consisted of repeats of palindromic DNA separated by unique spacer sequences, they were given the name clustered regularly interspaced short palindromic repeats, or CRISPR.

The function of these sequences remained an enigma for the next two decades, until Spanish researchers determined in 2005 that the unique spacer sequences match the DNA of viruses that prey on bacteria, leading to the hypothesis that CRISPR is an adaptive immune system. Two years later, in 2007, food scientists at Danisco studying the bacteria used to make yogurt confirmed experimentally that CRISPR is indeed a prokaryotic immune system.

How Does the CRISPR System Work?

CRISPR sequences are a repository of past infections. They contain snippets of dangerous viruses, which allow a cell to recognize those sequences and mount a successful defense against the next infection. Close by the CRISPR sequences are genes encoding for Cas (CRISPR-associated) enzymes, which form the second part of this defense mechanism. CRISPR sequences check for the presence of foreign genetic material, and Cas proteins carry out the work of destroying it.

In order to accomplish this task, a cell transcribes the spacer sequences into RNA molecules. These form a complex with a Cas enzyme, which drifts through the cell until it comes into contact with genetic material that matches the RNA. When this occurs, the CRISPR RNA binds tightly to the matching DNA, allowing the Cas enzyme to precisely cut the foreign DNA, thus preventing the virus from replicating. After successfully killing off an invading virus, other proteins cut up pieces of the viral DNA and store them as CRISPR spacer sequences to prepare for future infections.

Scientists Have Modified CRISPR into a Precise Genome-Editing Tool

One of the most well known Cas proteins is the nuclease Cas9, a DNA-slicing enzyme that comes from the bacteria that causes strep throat. In 2012, Jennifer Doudna and Emmanuelle Charpentier created a programmable CRISPR/Cas9 system with “considerable potential”. They simplified the system down to the Cas9 nuclease and a synthetic guide RNA, which allows it to precisely cut and paste pieces of DNA into the genome. Shortly thereafter, Feng Zhang and George Church successfully adapted this system for genome editing in eukaryotic cells.

In the years since, scientists have raced to improve upon this system to make it safer, more efficient, and more predictable. In 2012, there were just 126 papers published on CRISPR. By 2016, there were over 2000 publications. Startup companies founded by the key researchers have received approximately half a billion dollars in venture capital funding, and the technology has garnered considerable interest from the largest players in biotechnology, pharmaceuticals, and agriculture.

Recent Years Have Seen a Long Line of CRISPR “Firsts”

Researchers have used CRISPR to correct the sickle cell mutation in the cells that eventually turn into red blood cells, fix mutations in the blood stem cells of patients with a rare immunodeficiency disorder, and prevent HIV infection in human cells. One major application in biomedical research is to create mice with multiple specific mutations within a single generation, dramatically reducing the time and effort required to generate new disease models.

Last year, DuPont released its first commercial agricultural product developed through CRISPR-enabled advanced breeding technologies (which does not fall under current regulations for genetically modified organisms). Research into other CRISPR-modified plants and animals is proceeding rapidly, with notable examples including heritable targeted mutagenesis in tomato plants and gene-edited pigs that are resistant to the most economically important swine disease.

Technical Hurdles and Ethical Challenges Remain

One of the key technical challenges facing more widespread applications of CRISPR are off-target effects—undesired mutations that occur within unknown parts of the genome that limit our ability to make precise changes to the genome. In terms of therapeutic applications, targeting and delivery of the CRISPR machinery remains an important hurdle to overcome, particularly in solid tissues.

Aside from technical barriers, controversy remains about how to apply CRISPR outside of the laboratory. One well-known example is the modification of human cells. Chinese scientists have applied the technique to nonviable human embryos, and even though the results were not successful (due to unintended off-target mutations), prominent researchers have called for a moratorium on germline editing in order to hammer out the safety and ethics issues. However, the National Academy of Sciences and the National Academy of Medicine recently issued a statement that heritable human genome editing could, under stringent oversight, be allowed. There has been considerably less controversy around editing of somatic cells, with the first CRISPR clinical trial approved last year (to help augment cancer T-cell therapies).

With the closure of a recent high-stakes patent battle, which found no interference between patents from UC Berkeley and the Broad Institute, there may be more certainty about how to proceed with commercialization of the technology. And although many challenges remain—technical, regulatory, and ethical—we are rapidly overcoming them (with respect to the ethical concerns, some would argue too quickly).

Where Will CRISPR Take Us?

It is not unreasonable to start thinking about the next generation of applications. We could edit crops to make them tastier, more nutritious, and more robust to environmental stresses. We could use CRISPR-based therapeutics to correct the mutations responsible for genetic diseases such as Huntington’s disease or cystic fibrosis. We could create a powerful new generation of antibiotics and antivirals, and even use “gene drive” to modify the genome of an entire species (for example, malaria-carrying mosquitoes). And yes, we could potentially use this technology to select for desired traits in humans.

According to CRISPR pioneers Rodolphe Barrangou and Jennifer Doudna,

“beyond clinical applications this disruptive technology is on the brink of transforming agriculture, livestock breeding, food manufacturing and the biotech industries, with uses in plants, animals, bacteria, yeast and fungi for trait enhancement, breeding and fermentation improvements… It will not only serve as a fundamental component of the biologist’s toolkit but could also affect almost every aspect of life, and provide inspiration for future technological breakthroughs.”

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