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Figure on left reprinted with permission from Chemogenomic Profiling of Endogenous PARK2 Expression Using a Genome-Edited Coincidence Reporter. ACS Chemical Biology, May 1. Copyright 2015 American Chemical Society.

Gene Editing Technology with CRISPR-Cas9: What Life Sciences Researchers Need to Know

"I remember the first time I heard of CRISPR—reading the summary of a paper published in Science by Jennifer Doudna of the University of California, Berkeley and colleagues. It was one of those 'Oh my' moments. I saw the power and potential of the technology for functional genomics, and knew immediately that it was something I needed to be doing," says John Doench, Ph.D., a research scientist in the Broad Institute's RNAi Platform.


Samuel Hasson, Ph.D., principal investigator, Pfizer Neuroscience, also saw the power of CRISPR-Cas9 technology when, as a pharmacology research fellow, he was working to identify new drug targets for Parkinson's disease at the U.S. National Institute of Neurological Disorders and Stroke and Center for Translational Therapeutics. "We brought it into the lab just as I was transitioning to Pfizer, and what we saw was amazing. I'm glad to be continuing to work with the technology in my current job."

First experimentally characterized in 2007, the CRISPR (clustered regularly interspaced short palindromic repeats) system is derived from a bacterial immune system. "Essentially, the system uses RNA to cut up DNA," Doench explains. "To import it into your organism, you need just two things: the cas9 protein and an RNA to program it. The result is an RNA-guided DNA endonuclease that can be used to cut any DNA sequence in any organism of interest. Various approaches can be used to optimize single guide RNAs (sgRNAs) to maximize on-target activity and minimize off-target activity."

"Simply put, CRISPR has the potential to transform our ability to do biomedical research," says Hasson. "It extends the reach of what we can achieve with genetic manipulation, particularly in mammalian systems."

Hasson and Doench will share their enthusiasm and expertise in the use of the CRISPR-Cas9 system with participants in a new SLAS2016 Short Course, Gene Editing for Drug Discovery.

Editor's note: Innovations in gene editing using CRISPR, RNAi and other technologies can be found in the September 2015 special issue of the Journal of Biomolecular Screening (JBS) on Screening by RNAi and Precise Genome Editing Technologies by guest editors Marc Bickle, Hakim Djaballah and Lorenz Martin Mayr.

Why is gene-editing technology important for drug discovery right now?

John Doench (JD): Fifteen years ago we had the map of the human genome, but knowing all the "A"s, "C"s, "G"s and "T"s doesn't tell us anything about gene function. In order to understand how disease states are caused by gene dysfunction, we need to know what specific genes do in a lot of different cell types and contexts throughout the human body. Geneticists have been breaking genes, turning them off and learning what they do for 100 years. But classical genetics does this in a reverse manner, starting with a mutation and then figuring out what the gene is. With the publication of the human genome, we could say, 'Here's a gene, we know what the sequence is, what does it do?'

RNA interference (RNAi) was the first technology that allowed us to do this. It's a robust technique that opened doors that had been locked to biologists for decades. For example, in collaboration with the Duraisingh lab, we used RNAi technology to show that the malaria parasite, Plasmodium falciparum, uses the cell surface receptor CD55 to get inside red blood cells.

But CRISPR takes us to a whole new level by overcoming RNAi's two main limitations. The first is that, as the name implies, researchers can interfere only with RNA. By contrast, the CRISPR system allows us to edit DNA, so when a change is made using CRISPR technology, it's a permanent change. The second limitation is that RNAi has significant off-target problems; if you use multiple reagents meant to do the same thing, they don't always do the same thing, and that lack of reproducibility has been a problem. So far, it looks as though CRISPR is much more specific, which is important for both laboratory work and potential therapeutic applications.

Samuel Hasson (SH): With CRISPR, we can really get into the endogenous realm in R&D. We can endogenously tag a protein instead of having to overexpress it, and that can make a huge difference with respect to the results of our experiments. As John noted, we remove a layer of artifacts, and so we can make cleaner observations of phenotypic changes, and the results are clearer.

How are you using CRISPR-Cas9 in your own work?

JD: I love technology development. My job is to work with experts in infectious diseases, cancer biology, neurobiology and other areas who want to solve specific biological problems. I apply the latest genome perturbation tools, such as CRISPR, to their model system and, working together, we learn which genes are involved in their particular disease processes. For example, I've collaborated with researchers here at the Broad on various components of the immune system to learn about factors involved in influenza and norovirus infection. We've also used CRISPR technology in cancer biology to discover resistance genes, identify tumor suppressors and generally to determine the Achilles heels of various cancer cell lines. We're really applying it to as many types of biological problems as we can.

SH: During my postdoc, my main focus was on identifying new drug targets for Parkinson's disease related to mitochondrial quality control, and I've started in genome editing with TALEN technology. For example, with my co-mentors Jim Inglese and Richard Youle at the U.S. National Institutes of Health, I used genome editing (integrating a coincidence reporter gene into the PARK2 gene locus of a neuroblastoma-derived cell line) to really cut down on false positives in reporter gene-based small molecule screening. The approach led to the development of a quantitative high-throughput screening (qHTS) assay that can detect subtle compound-mediated increases in endogenous PARK2 expression, which could have a neuroprotective effect.

What other diseases might the technology be applied to?

SH: Right now, I am excited about immuno-oncology. It will take a while to work out the in vivo delivery side and make sure safety concerns are allayed, but I think immuno-oncology is where genome editing will make the most immediate impact.

JD: I think the first successful CRISPR applications will be in diseases where we can take cells out of the body, manipulate them and put them back in. This certainly is the case for the hematopoietic system: we can take out stem cells, modify them and put them right back into the same patient, where they can essentially repopulate the whole blood system. An example would be taking someone who has HIV and modifying their cells so they no longer express the CCR5 gene (people with a deletion in CCR5 are naturally resistant to HIV). Another example is sickle cell anemia, which is caused by a single mutation that could be fixed by taking out cells and editing in a change. That's the other real benefit to CRISPR technology relative to small molecule- or antibody-based therapy: it fixes the DNA, and so it can be a cure; there's no need to take a small molecule or make lifestyle changes for the rest of your life. You do the gene editing once, and you're done.

What are the challenges of CRISPR technology?

JD: One of the technical challenges is that cas9 is a big protein, and can't easily be applied to all cell types—especially those that are difficult to manipulate, like primary cells—using standard nucleic acid delivery methods. It's a technical challenge, but it needs to be solved in order to make CRISPR technology applicable to all model systems.

SH: Even though a lot of researchers are talking about CRISPR and more and more are doing it, I believe there are still activation barriers. People are holding onto their older methodologies and not moving into CRISPR as quickly as they could be. I'd like the scientific community to understand that CRISPR isn't a fad that's here today, gone tomorrow. It really is a major game changer for biomedical research.

Editor's note: In the JBS Special Issue on Screening by RNAi and Precise Genome Editing Technologies, Mark Wade authored a review entitled "High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges." He observes that while CRISPR offers "enormous potential" for high-throughput functional genomics studies, "the decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more 'mature' next-generation systems."

What about ethical concerns?

JD: We need to make a critical distinction between using CRISPR to modify somatic cells and using the technology to edit human germline cells, as was done recently. We can already screen for germline-related conditions with pre-implantation genetics. If someone wants to manipulate cells ex vivo, it's simply a matter of screening for the ones that don't carry the mutation and implanting those. There's no need to edit anything. IVF (in vitro fertilization) and PGD (pre-implantation genetic diagnosis) is something people have been doing for a decade, and there's nothing controversial about it.

While the concerns have been much discussed, 'designer babies' or 'fixing' traits such as intelligence or height are not within reach now, and may not be for a very long time. These phenotypes are the combination of so many different alleles, and hundreds of different single nucleotide polymorphisms, and we don't nearly understand the complex relationship among all these differences. Further, even if we did understand these networks of interactions perfectly, the multiplexing of edits that would be necessary is simply not feasible. It would involve editing not just a single base of DNA, but hundreds. What's the point of doing that?

SH: Just as with gene therapy, we need to be cautious with CRISPR and make sure that we're using appropriate applications of the technology. As discussed above, it's clear that CRISPR has promise for infectious diseases, immuno-oncology and a whole range of other conditions. We've already seen efforts to remove pathogenic mitochondria DNA from eggs and replace it with donor mitochondria to reduce the debilitating mitochondrial heteroplasmies that can cause serious juvenile diseases. But with CRISPR, people are getting ahead of themselves in terms of what it can do and how quickly it might change things. I personally believe human genetic engineering is something that's off in la-la land at this point. Right now, we need to avoid getting sidetracked and refocus on how the technology is changing researchers' ability to do in vitro and in vivo experiments and how this can improve the quality of the science and accelerate the pace of drug discovery.

What can the SLAS2016 Short Course offer researchers working in that regard?

JD: The CRISPR field has exploded in the last year or so. There's so much literature out there now that even if you're in the field, staying on top of it all is a challenge. If you're outside the field and thinking about getting into it, it's almost impossible to know where to start. One major goal of the course is to provide guidance to those people who may have worked with RNAi in the past, for example, and who have a model system but are not sure how to begin with CRISPR technology because there are so many options.

Cas9 is a remarkably programmable RNA-guided, DNA-binding protein. In addition to cutting DNA, we can tether things like transcriptional activators or repressors of epigenetic modifiers to it and use those as different ways of perturbing cells. So we will help participants understand that there's not just a single type of activity they can get with CRISPR-Cas9, but a whole suite of activities, with more to come. We want to lay out the various tasks that can be accomplished so people can go back to the lab and say, 'I know where I'm going to start with my experiment.'

SH: I'm excited because all the types of audiences that SLAS targets and attracts—assay developers, drug discovery researchers, academic tool developers, technology developers—will get a frank overview of CRISPR-Cas9 technology as well specific, practical ways to use it. And that will help overcome some of the activation barriers I spoke about earlier.

I grew up in the scientific generation that was told over and over how big a change PCR made to molecular biology. It's amazing to be part of another tectonic shift in our toolbox for both in vitro and in vivo work. Using CRISPR, our lab generated a new mouse model in less than three months. It was fantastic to see that this genome editing technique is usable, reproducible and applicable to so many areas, now and in the future.

SLAS2016 Short Course: Gene Editing for Drug Discovery

On Saturday, January 23, Doench and Hasson's course will provide a step-by-step approach to gene editing using CRISPR-Cas9 technology, including case studies from their respective laboratories and comparisons with other genome engineering technologies. Participants will learn about the technology's use in screening applications, with a focus on optimizing on-target activity and minimizing off-target effects. Future challenges and opportunities will also be discussed.

September 4, 2015