The recent emergence of easily accessible CRISPR-Cas9 technologies is enabling nearly unlimited opportunities for genome editing. Apart from its potential as a therapeutic tool, the system is currently spurring a revolution in drug discovery.

“The targets we’re finding with CRISPR-Cas9 are going to guide the drugs coming out in the 2020s,” said Jon Moore, CSO of Horizon Discovery, at a recent event in the UK. Only shortly after the first publication on the new genome engineering system in late 2012, the gene editing company and CRO started to recognize the potential of the new technology.

“Around 2013 we started getting interested in CRISPR-Cas9 (…) and over the next year and a half we went from predominantly generating models using AAV to almost exclusively using CRISPR-Cas9,” Chris Lowe, Head of Research Operations at Horizon, told us. Today, the company uses CRISPR across all of its platforms from engineering customized cell lines or animal models to performing functional screens. “We can generate hundreds of knock-out models a month on a rolling platform. And that’s really only possible because of the CRISPR-Cas9 technology. It’s pretty much all pervasive,” commented Chris.

To date, most of the attention on CRISPR has revolved around its potential as a therapeutic tool and the possibilities of engineering human embryos, crops or life stock. However, it seems like the real revolution right now is taking place in the lab. In 2015 alone, the scientific community published 1,185 publications (corresponding to 3 publications a day!) on the new gene editing system, and scientists have hacked the system to do far more than just cut DNA. CRISPR appears to be emerging as a key tool for drug discovery ranging from target identification and validation to preclinical testing.

CRISPR-Gene Editing

RNA-based Gene Editing

RNA-guided Cas9 nucleases, which are derived from microbial adaptive immune systems, are enabling fast and accurate alterations of genomic information in mammalian model systems, including human tissues. While genome editing tools are not entirely new, Chris told us that “the benefit of CRISPR really is in the speed and ease with which you can create the reagents necessary to perform gene editing,” thereby overcoming many limitations of its predecessors such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs).

Cas9 makes cuts at specific locations along the DNA with help from a short stretch of “guide RNA” that targets the Cas9 endonuclease to a specific site. By simply changing the guide RNA sequence, Cas9 can be directed to any site within the genome. The synthesis of such short pieces of RNA is way simpler than having to engineer a whole protein to direct it towards a specific DNA sequence.

The resulting double-strand break is then repaired by the cell’s error-prone DNA repair machinery. That alone is usually enough to knock-out the gene of interest and allows scientists to study what happens to cells or organisms when the protein or gene is shut off. Alternatively, the scientist can provide a piece of new DNA, maybe a new gene, which is then built in at the target site.


The RNA-guided Cas9 nuclease.

Screening for New Drug Targets

CRISPR gives scientists the opportunity to engineer and study virtually all cell types and it has become common practice around the globe. In fact, as the system is incredibly fast and cost-effective, it has enabled scientists, for the first time, to conduct high-throughput knock-out screens to speed up target discovery.

Using retroviral libraries of guide RNAs that target every single gene within the genome, CRISPR can be used to generate thousands of different cell lines at once, each containing a different guide RNA that targets a particular gene.

Principle setup of a CRISPR screen.

Feng Zhang’s lab, the first lab that used CRISPR to engineer human cells, made use of such genome-wide screens to address treatment resistance to melanoma. BRAF V600E is a common cancer mutation that is treated by the FDA–approved drug vemurafenib. Yet, the rapidly mutating cancer cells quickly become resistant, and by 24 weeks of treatment, the tumors return.

“We thought this might be an opportunity for us to apply a genome-scale library to see what are the genes—when you either turn them on or turn them off—that would render the tumor cell resistant to vemurafenib,” Zhang explained in an article in The Scientist.

Apart from identifying genes that make cells resistant to specific drugs, researchers are using the system to screen for genes that are essential to the cancer cells, but not normal cells — a state referred to as synthetic lethality. Others are using CRISPR screens to search for survival factors of pathogens such as the Zika and Dengue viruses.

Although RNA interference-based screens were widely used before CRISPR, the new system has considerable advantages. Most significantly, gene editing will lead to the complete inactivation of a target, compared to the incomplete knockdown seen with RNA interference (RNAi). In addition, confounding off-target effects of siRNA molecules are widely reported. As Chris told us, “we are seeing much greater reproducibility than what we’ve seen using RNAi over the years. So that’s a big element that’s driving the adoption of the CRISPR screening technique as a complementary technique to the siRNA approaches.”

CRISPR-Tailored Disease Models

A key to successful drug development is the availability of suitable model systems to make early drug development decisions. As Friedhelm Bladt, Director of Biomarker Strategy at Bayer, told us, “One limitation in drug development is that you test your efficacy in mouse models, sometimes in rats. But these animals react very differently from a human being and they are in some aspects much more robust than human beings would be.”

Generating a new disease model used to be a laborious and expensive task limited to a few species that came with a good tool kit for genetic manipulation. “CRISPR now allows us to generate much better animal models that really reflect the human situation,” commented Friedhelm.

Today, CRISPR has been used to engineer a wide range of species including rats, dogs and cynomolgous monkeys, which are all commonly used during preclinical drug discovery. Others are using it to engineer the genome of ferrets, in order to modify their susceptibility to flu infections. These animals are much better suited as influenza transmission models, due to the fact that unlike mice, ferrets sneeze when infected.

Another major advantage is that CRISPR allows tweaking more than one gene at a time, taking into account that most human diseases are not monogenic. “Tumors, for example, are very heterogeneous and you usually have a lot of different types of mutations as well as differences within the tumor. Modeling that is a huge challenge in animal models,” explained Friedhelm. “With CRISPR we are able to really introduce a set of mutations or potentially even introduce some heterogeneity in the tumors.”

Creating a mouse model with multiple mutations used to take years due to lenghty backcrossing, costing about $20,000 per mutation. With CRISPR, this time has been reduced to months or even weeks.

CRISRP-Animal Models

Hacking CRISPR

Apart from serving as a gene editing tool, CRISPR has already been hacked to do much more than that. As Chris explained: “I see the CRISPR system not so much as an editing tool but more as a targeting system. It allows us to precisely target tools to specific locations in the genome – and this ability is challenging our imagination, allowing the investigation of much more subtle effects on the genome compared to the fairly blunt technique that was brought out a couple of years ago where you just damage the DNA and let it repair.”

When the group of Jonathan Weissman at the University of California, San Francisco (UCSF) got hold of CRISPR, the first thing they did was to break the scissors, he explains in a recent Nature interview. The group mutated the Cas9 protein so that it still bound to the DNA but no longer cut it, allowing the team to turn off genes without changing the DNA sequence.

Then they tethered Cas9 to a protein that activates gene expression. They now had a simple system available that allowed them to turn genes either on or off at their will. Others are using CRISPR to make more subtle modifications to the DNA: by coupling CRISPR to epigenetic modifiers such as histone acetylases, scientists are able to study the direct effect of epigenetic marks, providing a straightforward tool to study how epigenetics can drive disease. “These types of alterations can be modified with CRISPR in a much more selective way than it was possible in the past,” explained Friedhelm. “And there are many more potential applications – people have just started to discover these.”


They Are All Using CRISPR

Since its appearance in 2012, CRISPR has given rise to a massive number of new tools that are impacting the entire drug discovery process. The system is redefining what’s possible in R&D, which is why many biotech and pharma companies have started integrating the technology into their R&D programs.

Novartis recently entered a partnership with Jennifer Doudna’s Caribou Biosciences to access Caribou’s CRISPR drug screening and validation technologies, while AstraZeneca signed up for four research collaborations to use CRISPR across its entire drug discovery platform. Similarly, German Evotec recently teamed up with Merck to access its CRISPR libraries that are based on a license from the Broad Institute.

As CRISPR Therapeutic’s CEO Rodger Novak told us at our last Refresh Event, “There is probably no larger biotech or pharma company out there anymore, who have their own R&D, who are not using CRISPR. They are all using CRISPR in their labs. It’s a very powerful technology, not only for human therapeutics.”

Images via / CHORNYI SERHII / Perception7 / unoL;;


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  • KapnKbeng

    Excellent scientific breakthrough.

  • les garson

    anyone looking at CRISPR technology to turn on and off genes responsible for state of anesthesia-we know certain anesthetic agents target specific receptors in brain (NMDA for example).