A breakthrough in genetic modification that promises to revolutionise drug discovery and could eventually underpin an entirely new way of treating disease has won this year’s Dr Paul Janssen Award for Biomedical Research.
The award, which celebrates research with the potential to go from ‘bench to bedside’, has been given this year to Drs Emmanuelle Charpentier and Jennifer Doudna in recognition of their discovery of the CRISPR-Cas9 method of genetic editing, which has made it possible to make exquisitely detailed and precise alterations to DNA sequences on demand.
Originally discovered in bacteria as a defence mechanism against pathogens, CRISP-Cas9 is already emerging as a powerful laboratory tool that promises to accelerate many different types of research, and is “considered one of the most significant breakthroughs in molecular biology in the past decade,” according to Paul Stoffels, chief scientific officer of Johnson & Johnson (J&J), which created the award to honour the legacy of Dr Janssen.
Speaking with PME, Charpentier (pictured right), who runs labs at the Hannover medical School and Helmholtz Centre for Infection Research (HZI) in Germany and Umea University in Sweden, described the discovery of the mechanism, which utilises a duplex RNA (tracrRNA:crRNA) to guide an enzyme (Cas9) to cleave DNA in a site-specific manner (see inset box).
“The tool consists of a single RNA that can be programmed to guide the enzyme to modify any sequence of DNA of interest in the genomes of any cell and organism,” she said.
Put simply, the technique introduces a break in a specific place within DNA and triggers a self-repair mechanism. However, instead of restoring the original sequence, CRISPR serves as a new template that can be used to change the sequence.
“The tool is efficient, easy-to-design and versatile and has been harnessed for gene deletion, correction, replacement, addition, expression modulation and modification,” said Charpentier. “It also allows manipulating more than one DNA site at once.”
Before this gene editing technology was available, gene manipulation in higher organisms was possible only in a limited number of higher organisms such as yeast or mice, according to Charpentier.
Winners of the Dr Paul Janssen Award for Biomedical Research
| Year | Name | Reason for award |
| 2014 | Jennifer Doudna and Emmanuelle Charpentier | The co-discovery of a new method for precisely manipulating genetic information in ways that should produce new insights in health and disease |
| 2013 | David Julius | Discover of the molecular mechanism that controls thermosensation and elucidation of the role this mechanism plays in the sensation of acute and inflammatory pain |
| 2012 | Victor Ambros and Gary Ruvkun | Discovery of microRNAs (miRNAs) as central regulators of gene expression and development |
| 2011 | Napoleone Ferrara | Research on angiogenesis, the process of new blood vessel formation |
| 2010 | Erik De Clercq and Anthony Fauci | Pioneering work in understanding and combating viral diseases, particularly HIV/AIDS |
| 2009 | Axel Ullrich | Pioneering work in applying molecular biology and molecular cloning to the discovery of protein therapeutics |
| 2008 | Marc Feldmann and Sir Ravinder Maini | The discovery of tumor necrosis factor-alpha as an effective therapeutic target for rheumatoid arthritis |
| 2006 | Craig Mello | The discovery of RNA interference and the elucidation of its biological functions |
The introduction of zinc-finger nucleases (ZFNs) and TALE nucleases (TALENs) in the genome engineering toolbox has already allowed gene manipulation in additional higher organisms such as human cells. These enzymes introduce double stranded DNA breaks in a genome in a site-specific manner but – in contrast to CRISPR-Cas9 – rely on protein-DNA recognition and so – for each new sequence to be targeted – a new enzyme needs to be engineered, which has time and cost implications.
“The difficulties of protein design, synthesis and validation inherent to these engineered nucleases remained a barrier to their widespread adoption for routine use,” said Charpentier. “In contrast, by the simplicity of its RNA programmability and design, CRISPR-Cas9 has now been adopted by the scientific community.”
The low cost, speed and ease of application of the technology has made it so successful, as any lab with biological expertise can deploy it. There are more than 600,000 mutations associated with cancer, for example, but only a few have been studied in depth using the earlier gene silencing methods and the new tool is accelerating that effort.
Scientists are already using it to perform genetic engineering in higher cells and organisms in order to understand the genetic codes and principles responsible for functions in cells and organisms, and to characterise diseases by linking genes to diseases. Meanwhile, CRISPRCas9 is also being developed as a tool for gene and drug screening and to validate biological and pharmacological tests and disease models used in drug development.
An RNA guide molecule is created that acts as a homing device, zeroing in on any target DNA sequence in a genome.
A nuclease enzyme called CAS9 is attached to the RNA guide to create the CRISPR-Cas9 construct.
A sequence on the guide RNA known as a spacer aligns with and binds to the target DNA sequence – alongside a highly conserved motif on DNA known as a Protospacer Adjacent Motif (PAM).
The CAS9 enzyme cuts both strands of the DNA double helix upstream of the PAM.
Once the DNA strand is cut, a cellular repair mechanism comes into play that can be manipulated, for example by introducing a stop codon to silence the sequence or inserting an alternative sequence to alter it.
The system is cheap and quick as only the spacer needs to be modified in order to target a new DNA sequence. It is also very small, which means it can be carried via a viral vector such as a lentivirus and be used to modify the genomes of living animals much more easily.
Bill Strohl, vice president head of the biotechnology center of excellence at Janssen R&D, explained how CRISPR-Cas9 is already becoming an indispensable tool at the company. “We are utilising gene editing technologies in a variety of ways ranging from enabling novel drug discovery to exploring gene therapy,” he told PME.
Preclinical target identification and validation, particularly for targets of small molecules, is a key application, he continued, while in many cases, specific in vitro and in vivo knockouts or substitution of a gene with a pertinent allele can help determine quickly the unique biology dictated by that gene.
“In a recent study using the newer gene editing methods, generation of specifi c genetically modified animals was achieved in four months, which is several times faster than possible with traditional procedures,” said Strohl. “These knockouts are already providing valuable information for screening monoclonal antibodies against a pertinent cell surface target.”
Similarly, Janssen has been able to make and test multiple gene variants in cell lines that can be used for in vitro screening to help determine which ‘hits’ are most physiologically relevant.
Therapeutic potential?
Clearly, much of the excitement surrounding gene editing lies in its use as a direct therapeutic tool for the treatment of human genetic disorders and other diseases such as viral infections. For example, it might be possible in time to modify an individual’s DNA so that it no longer codes for a key protein used by HIV to infect cells, while earlier this year scientists used gene editing techniques to cure adult laboratory mice of an inherited liver disease.
Clearly, there is however a long way to go before gene editing becomes a viable therapeutic approach, and a lot of work will have to be carried out on potential risks before the technique transfers from the lab to the clinic.
“Questions remaining to be answered before the technology could become a therapeutic modality include the improvement of homologous recombination rates by CRISPR-Cas9,” commented Charpentier.
Work will also have to be done to develop tools that can deliver CRISPR-Cas9 in targeted cells and tissues, and any associated safety issues must be considered, she added.
On the latter issue, gene editing researcher Feng Zhang of MIT in the US told the institute’s annual EmTech conference last month that “making any correction in the body is very challenging,” alluding to the unexpected hazards of gene therapy and specifically the tragic death in 1999 of Jesse Gelsinger, a teenage volunteer in one of the first clinical trials of the technology.
It is already known that CRISPR can sometimes change genes other than those intended as targets for editing, and of course that could lead to unwanted side effects.
Getting gene editing drugs into the cells of the body is also likely to be challenging, and scientists will have to explore a number of delivery approaches – for example modifying a patient’s cells ex vivo to correct a defect and then transplanting those cells back into the body, said Zhang.
Our goal is to go beyond simply treating diseases; to preventing, and potentially even curing them
Despite the expected challenges, there has been considerable interest in the CRISPR-Cas9 technology and applications in the pharmaceutical industry. Charpentier has co-founded CRISPR Therapeutics aiming to apply CRISPR-Cas9 in the medical fi eld notably for the treatment of severe human genetic disorders, while Doudna, Zhang and others have set up Editas Medicine.
Janssen meanwhile is “beginning to design therapeutic programmes using gene editing technology that go beyond established methods to address targets in diseases such as colorectal cancer, Alzheimer’s disease, inflammatory bowel disease and heart failure,” according to Strohl.
“Our goal is to go beyond simply treating diseases; to intercepting, preventing, and potentially even curing diseases, and the new gene editing technologies offer unprecedented opportunities to achieve these goals,” he said.
As this article went to press, Doudna’s research team revealed that they have programmed CRISP-Cas9 to recognise and interact with not only DNA but also RNA sequences, suggesting the technology could also be used to increase, decrease or block protein synthesis from messenger RNA and also serve as a tool to study intracellular processes.
