In Focus Cite This: ACS Chem. Biol. 2018, 13, 296−304
Frontiers in CRISPR Alyson G. Weidmann* ABSTRACT: CRISPR-based approaches to genetic engineering are progressing at a rapid pace and present exciting new avenues for science, medicine, and technology. Many of the most cutting-edge advances in genome engineering are encompassed in the Research Articles, Reviews, and Perspectives in this special issue, often with an eye toward future directions for the field. Yet, many questions remain at this new frontier. We asked over 100 CRISPR researchers, including our contributing authors, for their perspectives on some of the most pressing questions surrounding the future of genome engineering and the CRISPR-Cas platform, the challenges that lie ahead, and opportunities for chemists and chemical biologists to drive creative molecular solutions.
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EMMANUELLE CHARPENTIER
2. Which application areas will be impacted earliest by CRISPR, and where will CRISPR technologies have the most disruptive effect? Crop engineering with CRISPR-Cas9 can be realized much faster than medical applications because there are fewer ethical issues and regulatory challenges. There is a high interest in the field of agriculture to use the technology to make crops more resistant against the effects of climate change or to bring to market plants that need fewer amounts of fertilizers. Research has already come a long way here. While the EU has still not decided on regulations regarding CRISPR-engineered plants, CRISPRengineered mushrooms that do not become brown as fast as conventional mushrooms are already in supermarkets in the U.S.A. But of course, the CRISPR-Cas9 technology is also very promising in the medical field when it comes to the possible treatment of a number of serious genetic diseases. The technology is already largely used in research and development to accelerate the understanding of mechanisms of diseases, engineer more suitable and clinically relevant disease models, screen for new targets for therapeutics, and test drugs under development. It will take more time until we can see the technology being applied as a gene therapy for humans. After all, we are still in the early days of CRISPR research and need be sure that the right cells or cell types are targeted. The team at CRISPR Therapeutics, the company I cofounded with Rodger Novak and Shaun Foy, recently filed an application for clinical trials for the possible treatment of genetic blood disorders such as β-thalassemia and sickle cell disease, and I am very confident that we will see good results soonhopefully with trials for other diseases to follow. 3. What CRISPR applications do you think will be the first to reach the clinic? After we discovered the CRISPR-Cas9 mechanism and were able to harness it as a genetic tool, thousands of scientists around the world have started translating this technology into safe and effective therapies to treat serious human diseases for which there are currently no treatment options. I am truly amazed by the advancements that have been made only a few years after our discovery. In December, CRISPR Therapeutics, the company I cofounded with Rodger Novak, filed an application for clinical trials for CTX001 in the genetic blood disorders β-thalassemia and sickle
Reprinted by permission from Hallbauer & Fioretti (Braunschweig, Germany)
Prof. Dr. Emmanuelle Charpentier, Director of the Max Planck Institute for Infection Biology in Berlin, Germany, answered all of our questions. She is widely recognized as one of the pioneers in the revolutionary discovery of the molecular mechanisms underlying CRISPR-Cas systems. An expert in the regulatory mechanisms of bacterial infection and immunity, Prof. Charpentier and her laboratory uncovered the RNA-mediated pathways that regulate how prokaryotes deploy the CRISPR-Cas system as an immune defense. Her findings, which revealed how the Cas9 effector protein is directed to cut DNA at specific sequences, have helped pave the way for ground-breaking advances in genome engineering. 1. What are some of the persistent challenges in the field? I would not say that the persistent challenges of CRISPR-Cas research are any different from those encountered in other fields of microbiology or biology in general. From my side, for example, and from the very beginning, it was clear that we had to use a combined physiological, genetic, cellular, molecular, and biochemical approach to identify the components of the CRISPRCas9 system; understand how they work together to target DNA in bacteria; and then to develop the mechanism as a versatile genetic tool that would work in any cell and organism. Of course, this involves a lot of brainstorming and a methodological approach that combines theoretical and hypothetical frameworks and several back-up plans. © 2018 American Chemical Society
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focusing on Gram-positive bacterial human pathogens. We are interested in understanding how RNAs and proteins control cellular processes on the transcriptional, post-transcriptional, and post-translational level. We study regulatory RNAs and proteins in various biological pathways such as horizontal gene transfer, adaptation to stress, physiology, persistence, virulence, infection, and immunity. In particular, we do research on interference systems in the defense against genetic elements (CRISPR-Cas); on small regulatory RNAs that interfere with pathogenic processes; on protein quality control that regulates bacterial adaptation, physiology, and virulence; on basic principles of DNA replication and its role for life; and on bacterial and vesicular interactions with host innate immunity. It is our aim to better understand these mechanisms and generate new findings in basic science that can possibly be translated into new biotechnological and biomedical applications, e.g., genome editing tools or anti-infective strategies.
cell disease. If everything goes well, the trials should start in Europe in 2018. CTX001 is an investigational CRISPR geneedited autologous hematopoietic stem cell therapy, in which a patient’s hematopoietic stem cells are engineered to produce high levels of fetal hemoglobin (HbF; hemoglobin F) in red blood cells to alleviate transfusion requirements for β-thalassemia patients and painful and debilitating sickle crises for sickle cell patients. I sincerely hope that applications for other serious genetic diseases can follow soon. 4. As this technology matures and becomes more accessible, how do you think it will affect natural ecosystems? I am a bit concerned about the recent discussions of gene drive with the CRISPR-Cas9 technology. Gene drive describes experimental techniques which are supposed to push foreign genes into the chromosomes of wild populations with the aim to change the complete organisms in just a few generations. A prominent example is to target the female Anopheles mosquito which carries Plasmodium parasites that cause malaria. With the CRISPR technology, these changes can be realized very efficiently, especially in plants and insects that breed fast. But the method is ethically questionable because we still do not know if the genetic changes could affect other organisms or even entire ecosystems in a negative way. 5. Which applications are you most excited to see CRISPR technologies applied to? The CRISPR-Cas technology is extremely versatile. For this reason, it was adapted around the world where scientists are applying the technology for the basic understanding of mechanisms of life in a large diversity of cells and organisms and are working on potential future applications. I am very excited about the potential of CRISPR-Cas9 for the treatment of serious genetic diseases for which there is currently no cure but also for cancer or infectious diseases. This is one of the reasons I cofounded CRISPR Therapeutics. The CRISPR-Cas9 technology is also very attractive for agricultural research. Scientists are using it in crop engineering, because it can reduce the problems that appear randomly through conventional breeding. This, in turn, means time and cost savings but also more security. Also, it is possible to breed plants that are less receptive to parasites and fungi and more resistant against climate change. 6. What are your major ethical concerns surrounding CRISPR applications? Because it has such a simple design and is quite easy to use, the CRISPR-Cas9 technology is a very versatile and effective tool. The technology’s potential to be applied to basically any organism also brings along a lot of private interest. In the wrong hands, it clearly bears certain risks. Using CRISPR-Cas9 in human germlines is problematic, but some recent publications already show how far scientists have already come. There is a need for more discussions and international regulations about the potential risks of CRISPR-Cas9 as a gene-editing technology. As scientists, we also bear a certain responsibility: We need to make sure that appropriate safety and efficacy measures for any potential therapy that involve patients are taken and that any use of the technology that is ethically questionable is prohibited. Nevertheless, I think that it is crucial to go forward with the technology for research purposes because it gives us the opportunity to understand important mechanisms of life and diseases. 7. What is your research focused on today? In my lab, we continue to investigate the fundamental mechanisms of regulation in processes of infection and immunity,
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FENG ZHANG
Image credit: Justin Knight. Image courtesy of McGovern Institute for Brain Research at MIT.
We received in-depth responses also from Prof. Feng Zhang, Professor in Neuroscience at MIT and core institute member at the Broad Institute of MIT and Harvard. Prof. Zhang is also an investigator at the McGovern Institute for Brain Research at MIT and an associate professor at MIT, with joint appointments in the departments of Brain and Cognitive Sciences and Biological Engineering. A molecular biologist by training, he has exhibited the scope of the CRISPR-Cas genome engineering potential with applications in eukaryoticand even humancells. The tools developed in his laboratory leverage CRISPR methods to elucidate the genetics underlying many complex human diseases, including neurological disorders and cancer, and ultimately develop new therapeutic strategies for treatment. 1. What are the most exciting fundamental questions surrounding CRISPR biology? Two of the most exciting fundamental questions surrounding CRISPR biology are, (1) what are all of the different types of CRISPR systems that nature has created, and how they do they all work, and (2) how do these different types of CRISPR systems help bacteria defend against viruses, and how can we use this diversity? 2. What are some of the persistent challenges in the field? One of the persistent challenges facing the field of genome editing is the need to be able to deliver systems with all the elements needed to efficiently and precisely integrate a DNA template into the intended location of the genome. Current methodologies rely on the homologous recombination machineries in the cell, which are largely inactive in postmitotic cells. 297
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today, the one central theme that keeps on coming back to me is the importance of basic science research and mentoring students to nurture their inner curiosity and creativity so that they continue to explore life’s diversity. The work of all of those future scientists will undoubtedly reveal fascinating truths about the way life works and lead to new technologies that can continue to improve our lives and support the diversity that exists in our environment.
Versatile delivery methods are also needed to make it possible to introduce such genome editing technologies to different tissues in the body. 3. Which application areas will be impacted earliest by CRISPR, and where will CRISPR technologies have the most disruptive effect? CRISPR technology has already made a huge impact in scientific research, significantly accelerating the pace with which scientists can make genetic manipulations to cells and organisms to advance our knowledge about biology. Being able to distribute CRISPR-based tools broadly will continue to bring opportunities for laboratories around the world to undertake projects that would not have been possible without such shared reagents. In the future, application of CRISPR technology will likely have a huge impact on agriculture as well as diagnostics and treatment of diseases. 4. What CRISPR applications do you think will be the first to reach the clinic? The first therapeutic applications of the CRISPR technology will likely target tissues and organs with established delivery systems, such as the use of ex vivo genome editing to modify and return cells from an individual patient to treat thalassemia or cancer or the use of in vivo genome editing in the eye to treat blindness. 5. As this technology matures and becomes more accessible, how do you think it will affect natural ecosystems? We need to be thoughtful about how the CRISPR technology is applied in the context of environmental engineering or remediation, particularly paying attention to establishing control mechanisms and safety precautions. For example, the use of CRISPR-based gene drives needs to be carefully considered because of its potential impact on the environment. We also need to continue to explore methods and means to precisely control and inactivate unintended activity. 6. Which applications are you most excited to see CRISPR technologies applied to? It will be very exciting to see the continued realization of CRISPR-based therapeutics for treating genetic diseases. Using CRISPR-based tools to study the mechanism of simple monogenic and complex polygenic diseases will lead to novel treatments that have the potential to correct the underlying cause of genetic diseases and provide cures. 7. Are there specific areas of CRISPR research that could benefit from chemical biology? Chemical biology approaches have already been applied extensively to further optimize and enhance the function and efficacy of CRISPR technologies. This includes the development of inducible and controlled CRISPR systems, chemical modification of guide RNAs, and development of delivery technologies. 8. What are your major ethical concerns surrounding CRISPR applications? I think the use of CRISPR-based gene drives is particularly complex from the ethical perspective. Because organisms carrying gene drives have the potential to affect entire populations and eco-systems, the long-term and systemic effect of gene drives on the environment is difficult to predict. We need to be very cautious when advancing gene drives toward field trials. 9. What have you learned from your work with CRISPR systems and technology? When I step back and think about all of the work over the last many decades to discover and understand the natural CRISPR systems, and to engineer CRISPR-Cas9, and now also Cpf1 and Cas13, into the suite of powerful CRISPR technologies we have
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GEORGE CHURCH
Image courtesy of Wyss Institute at Harvard University.
ACS Chemical Biology also received responses from Prof. George Church, Professor of Genetics at Harvard Medical School and Health Sciences and Technology at Harvard and MIT. He is a founding member of the Wyss Institute for Biologically Inspired Engineering at Harvard. With expertise in synthetic biology, Prof. Church has pioneered multiplex precision genome engineering technologies in microbial and human stem cells, co-opting sequence-specific nucleases and homologous recombination (MAGE and CRISPR). His laboratory has also established CRISPR platforms for engineered organs and gene drives in wild species. 1. What are the most exciting fundamental questions surrounding CRISPR biology? The revolution is not really about CRISPR but about gene editing using many methods: ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), MAGE (multiplex automated genome engineering), Red/ET, integrases, et cetera. Also, genome reading is part of the revolution. 2. What are the some of the persistent challenges in the field? It would be nice to have higher efficiency delivery in vivo and higher fraction of precise editing rather than merely gene disruption (aka NHEJ). 3. Which application areas will be impacted earliest by CRISPR, and where will CRISPR technologies have the most disruptive effect? Earliest: We’re already seeing application to HPV, hemoglobinopathies, CAR-T treatment of cancers, et cetera. Among the most positively disruptive: Gene drives to eliminate malaria, Lyme disease, and invasive species. Gene therapies already constitute the most expensive drugs in history and may stay that way for quite some time. Gene drives, in contrast, may be even less expensive than the current most cost-effective medicine and vaccines. Vaccines to smallpox and polio required medical personnel to find every person in even remote villages, while gene drives might be able to leverage immunization of populations. 298
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of proteins has been discovered that target and cut RNA. I am certain that there is additional diversity in nature still to be discovered which can expand the use of CRISPR in genome editing. Jennifer A. Mitchell, University of Toronto The discovery and characterization of novel immune system types remains one of the most exciting areas of CRISPR-Cas biology. As recent studies have demonstrated, bacteria have evolved a remarkably diverse suite of CRISPR-associated enzymes to combat foreign genetic elements, and there’s still plenty of work left to unravel their mechanisms of action. Understanding the molecular requirements and biochemical functions of novel Cas effectors will open up new tools, like the development of Cas13 for nucleic acid detection, as well as orthogonal approaches for conventional gene-editing applications. Equally exciting is the discovery by Sorek and colleagues of entirely new (non-CRISPR) antiphage defense systems, which highlights the sheer diversity of microbial immune systems and serves as a reminder of the many open research questions that remain. Samuel H. Sternberg, Columbia University Medical Center Is there a universal connection between CRISPR-Cas immunity and programmed cell death/dormancy induction? The existence of such a connection, with altruistic suicide/shutdown being a measure of last resort, seems plausible given that numerous CRISPR-Cas loci encode toxins, and the type VI effectors are toxins themselves. However, definitive experiments are still lacking. Eugene V. Koonin, National Center for Biotechnology Information I think the most exciting question is what other CRISPRrelated systems are out there that may potentially be harnessed for genome engineering or mammalian synthetic biology. By and large, the most popular enzyme that is used today is Cas9 from Streptococcus pyogenes. Of course, we also see a few other Cas9 or Cpf1 enzymes from other bacteria being utilized, but there is just so much more to be explored. This includes anti-CRISPR systems from bacteriophages and other programmable nonCRISPR-based systems. Meng How Tan, Nanyang Technological University 2. What are the some of the persistent challenges in the field? One of them is to know the reason for the absence of CRISPRlike systems in eukaryotic cells and in many prokaryotes, even in strains of a given prokaryotic species while other isolates of the same species carry active CRISPR systems. Another persistent challenge is to understand the molecular determinants that govern the selection of CRISPR-spacer precursors. This advance would explain why certain sequences are integrated as spacers in the CRISPR arrays more frequently than others are and why there is a preference for particular DNA replicons as spacer donors, if compared to apparently equivalent molecules. Francis Mojica, Universidad de Alicante It is considered that CRISPR/Cas9 system-based gene therapy will be an effective method for genetic disorder treatments. However, the spatiotemporal resolution of current gene therapy technology is not high enough. It is not easy to avoid “leaky” Cas9 expression into the nontarget cells and organs, which in turn can lead to unexpected adverse reactions. In addition, it is difficult to stop the Cas9 expression at the intended timing if and when the genetic mutation is already corrected. Thus, there is the need for a new technology to spatiotemporally control a CRISPR/Cas9 system-based gene therapy. Kazuo Takayama, Osaka University
4. As this technology matures and becomes more accessible, how do you think it will affect natural ecosystems? Gene drives in mosquito vectors (aimed at malaria parasites) and in mice vectors (aimed at Lyme disease bacteria and ticks) might have very little impact on ecosystems, based on what is known about those ecosystems so far. Drives directed at invasive species could potentially restore endangered ecosystems, especially on islands with rare birds and reptiles. 5. Which applications are you most excited to see CRISPR technologies applied to? I’m most excited about genome editing that involves a large number of changes per genome, for example, 50 to 100 in pigs (for xenotransplantation) and elephants (to aid arctic carbon sequestration), and thousands of mutations to make genomes resistant to all viruses. 6. What are your major ethical concerns surrounding CRISPR applications? I’m concerned that people may suffer if safety tests are delayed a half million people per year die from malaria, a million per year from nutritional deficiencies, and 5 million per year from serious Mendelian diseases. ozens of CRISPR researchers around the globe offered their insights to our questions, with expertise spanning chemical biology, molecular biology, and microbiology. Here, we present their perspectives on the challenges, applications, and future directions of CRISPR-Cas. 1. What are the most exciting fundamental questions surrounding CRISPR biology? Cas enzymes (and even new base editors) initiate DNA repair pathways at specific locations. But how do those pathways really work at a detailed level in human cells? Thanks to decades of hard work, we have a great overall picture of DNA repair. But if we ever want to have truly predictable gene editing, we need even greater fundamental understanding of how DNA repair works in all manner of human cells. We need to be able to predict exactly how each lesion will be repaired in almost every cell type. Answering this grand challenge will yield fascinating basic knowledge, plus turn gene editing from an empiric process of trial and error into a predictable tool. Jacob E. Corn, UC Berkeley The mechanisms and extent of self vs nonself discrimination. Some of the CRISPR-Cas systems appear to prefer actively replicating and/or actively transcribed DNA. However, these are weak discrimination mechanisms. Other systems appear to be altogether wasteful, with a large fraction of cells being killed by autoimmunity. Are CRISPR-Cas systems indeed indiscriminate, which obviously makes them enormously costly, or are we missing some key aspects of their function that result in drastic reduction of the cost? Eugene V. Koonin, National Center for Biotechnology Information How on earth does Cas9 scan the genome (so tightly coiled and compacted inside the nucleus) and find its target so efficiently? This is like “trying to find one specific spot of a ∼1 cm long region of spaghetti in a gigantic pile of spaghetti stuffed inside a large room.” Channabasavaiah B. Gurumurthy, University of Nebraska Medical Center Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a form of adaptive immunity that evolved in bacteria and archaea to protect their genome from foreign nucleic acids. What has been so exciting about the past few years is discovering the many different ways that CRISPR has evolved in different species. The most commonly used CRISPR protein, Cas9 from Streptococcus pyogenes, creates a double strand break in DNA at a precise location, guided by an RNA. More recently, a new family
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pathway, which can introduce insertions or deletions of a few base pairs. These types of changes disrupt gene function when they introduce a frame shift in the coding region of a gene. The frequency of introducing a specific change, for example, correcting a diseases causing mutation, can be improved by using approaches to bias the repair pathway toward homology directed repair rather than nonhomologous end joining; however, these approaches remain less efficient than would be required to correct disease causing mutations in patient tissues. In my lab, we select clones of cells that have the modifications we want to study on either both alleles or only one allele. This allows us to study fundamental mechanisms of gene regulation in a controlled way and has improved our understanding of these mechanisms in only a few years of using these techniques (Moorthy et al., 2017; Zhou et al., 2014). In a clinical context, a selection approach only works for cells that can be modified in the lab and then given back to a patient. For example, selection approaches to ensure all cells have received a specific DNA modification can be used when cells of the hematopoietic system are modified, as these can be removed from a patient, modified, selected, and then introduced back into the patient. When targeting genome editing to solid tissues, for example, the liver, heart, or brain, alternative approaches need to be developed to improve efficiency. One new technique that has great promise for precise editing to correct a disease mutation is direct programmable base editing (Gaudelli et al., 2017; Cox et al., 2017). Jennifer A. Mitchell, University of Toronto There are challenges surrounding the “form, quantity, and delivery modes” of CRISPR components to target cells, particularly for targeting large number of cells in a somatic tissue. Additional challenges include developing methods to insert large DNA cassettes into genomes, efficiently and seamlessly. Channabasavaiah B. Gurumurthy, University of Nebraska Medical Center Some of the challenges that were identified earlier on still remain today. First, the efficiency of precision genome engineering is still not good enough in general. Although new techniques like base editing hold promise to correct many diseaseassociated point mutations, their overall utility has not been rigorously evaluated and is confined to specific DNA edits (such as C-to-T or A-to-G conversions). Second, the large size of Cas proteins hampers in vivo delivery. It is challenging to fuse effector domains to a catalytically dead Cas protein and package the entire fusion construct into an AAV vector. Meng How Tan, Nanyang Technological University CRISPR research can be divided into two major areas of interest: applied sciences (gene editing) and basic research. While data sharing between the two groups is mutually beneficial, the persistent challenges are recognizably distinct. On the applied side, the major challenges include increasing the programmable repair of Cas9 induced DNA breaks for restoring function to mutant genes (i.e., targeted repair rather than targeted cleavage) and in situ delivery of the Cas9 nuclease to tissues where the repair needs to occur. Basic research on DNA repair and DNA delivery will have a lot to offer as this field moves toward treating a wide spectrum of genetic diseases. For scientists focused on understanding the molecular mechanisms of adaptive immunity, the task continues to grow as phylogenetic studies uncover a seemingly limitless expanse of new CRISPR systems. While the theme of CRISPR RNA-guided detection of invading nucleic acids continues to be the hallmark of this system, the step-by-step process that is necessary for integrating foreign DNA at the correct location and in the correct orientation remains the least
Knocking out sequences is currently pretty easy. But inserting and swapping new sequences is much more difficult. This is frustrating since knockout is a bit like RNAi version 2.0. Casbased gene knockout is better, faster, and cheaper, but it is not a totally new capability. Highly efficient sequence insertion and replacement would be hugely enabling for fundamental research, from determining the functional consequences of individual variation to cleanly testing mechanistic hypotheses. This capability would also enable a great many therapeutic applications, including reversing disease mutations and engineering cell therapies. Jacob E. Corn, UC Berkeley The ability of CRISPR technology to reliably perturb gene function without significant off-target effects has rapidly transformed the field of genetic screening in mammalian cells. Still, an ongoing challenge is the development of model systems that can be screened at scale while faithfully capturing core aspects of disease biology. Because genome-scale screens require large cell numbers, the technology has typically been limited to diseases in which the relevant cell type is known and can be cultured and edited. Cost-effective pooled screens have limited phenotypic readouts; typically, cells receiving a “hit” perturbation must either undergo a change in viability or alter the expression of a marker that can be sorted by flow cytometry. Arrayed screens, or singlecell RNA sequencing of pooled screens, allow for richer readouts, but such approaches are generally limited in scale due to cost. Here, opportunistic selection of candidate genes to test is a good way forward, although this requires vigilance to avoid confirmation bias. Cumulatively, these challenges have largely restricted the impact of CRISPR screening to cancer biology, infectious disease, and basic cellular processes, while leaving behind diseases with complex phenotypes that are more difficult to model in vitro, such as cardiovascular, autoimmune, and psychiatric disease. Innovation and optimization are thus required to execute screens at scale in these disease areas in vivo. Even after the Herculean task of establishing a faithful animal model, developing an assay that allows the screening of many perturbations per animal still requires that the phenotype of interest be reduced to a cell intrinsic one, such as altered expression or viability. Where an in vivo assay improves upon an in vitro one is its ability to capture interactions with other cell types, but this too presents a challenge: results are likely to be confounded if multiple cell types other than the disease-relevant ones receive the perturbation. Here, ongoing efforts to catalogue distinct cell types will enable both the better definition of the relevant diseased cell type and, in turn, the creation of better tools to limit the activity of Cas9 to only desired cells. Additionally, in vivo screens will require the continued development of tools, such as lentivirus and AAV vectors, to deliver perturbations to endogenous tissues. Finetuning each model system will likely be challenging, but the payoff is significant: where they have been applied already, CRISPR screens have proven to be a powerful method for discovering gene function and represent the most promising tool yet for uncovering the underlying genetic drivers of complex disease. Ruth E. Hanna and John G. Doench, Broad Institute of MIT and Harvard One of the most persistent challenges in the field of genome engineering is the inability to modify DNA to a specific sequence in a highly efficient way such that all cells in a population receive the same modification at both alleles. A challenge in genome editing is that the same modification to the genome does not occur in all cells in a population or on both alleles within the same cell. This is because the Cas9 protein introduces a double strand break that is repaired by the nonhomologous end joining 300
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well understood aspect of this process. Blake Wiedenhef t, Montana State University Through groundbreaking work over the past decade, we now face the possibility of curing many genetic diseases by “editing” out the mutations that cause them. Early barriers to gene editing were to do with the nucleases themselves. Subsequent efforts were devoted to figuring out where else they cut and reducing these so-called “off-target” effects. The advent of CRISPR/Cas9 has greatly accelerated our progress. If we can get the nuclease, a guide, and a replacement sequence into a cell, efficient and precise editing with near-perfect fidelity is now routine. But this is only a small part of the challenge: our tissues are complex and vast, dense with billions of target cells, not to mention a phalanx of immune cells devoted to eliminating any unexpected reagents, or potentially cells that were treated with these reagents. The greatest challenge, the challenge we face now, is delivery. There is a subset of organs that are readily accessible using current technology: the blood (via stem cell transplant), the liver, perhaps the kidney. Even there, challenges remain. For example, efficient sequence replacement in blood stem cells is a major challenge. How do we get these reagents to the cells of the brain? Heart? Lungs? Luckily, we can borrow from earlier work, much of it less-than-successful, from drug development. Antibody− drug conjugates, nanoparticles, bioconjugate chemistry, immune stealth, all these well-trod paths need to be adapted to gene editing systems to get the reagents where they need to go, safely and effectively. If these barriers to delivery can be met, a bright future comes into focus: any tissue, any mutation, in any patient. This may lead to a new barrier: target discovery. If any manipulation is possible, what do we do? Genetic target discovery is a difficult process. For some genetic diseases, the cause is clear. In sickle cell disease, for example, a single mutation is the sole root cause of the disease. But what about diseases with a genetic component, that are not strictly inherited? Alzheimer’s, Parkinson’s, adult-onset diabetes, and many others may have genetic “cures” that can be surmised on a personalized basis. The Herculean effort required to discover these curative genetic changes will itself be enabled by gene editing, through rapid generation of disease model cell lines. The future of gene editing is indeed bright. If these two barriers of delivery and target discovery can be overcome, it is a tool that could help transform the lives of all of us for the better. Mark DeWitt, Innovative Genomics Institute, UC Berkeley 3. Which application areas will be impacted earliest by CRISPR, and where will CRISPR technologies have the most disruptive effect? The excitement surrounding the CRISPR gene editing system is far reaching and contagious. This technology has captured the interest of such diverse fields of science ranging from biology to engineering. I feel that merging of the fundamental gene-targeting capacity of CRISPR with unique and innovative device designs to create CRISPR-powered diagnostics holds the greatest potential to make the most rapid and disruptive impact. The first obvious area of impact will be the medical field; however, as we continue to push the limits of the technology, alternative fields will emerge. Tara DeBoer, UC Berkeley CRISPR technologies have already had a huge cross-disciplinary impact on fundamental biological science. In the context of studying the function of mammalian genomes, CRISPR technology has allowed for genome-wide genetic screening and the genetic disruption of entire gene families in more rapid and accessible way than ever before. Jennifer A. Mitchell, University of Toronto
One area of disruption that CRISPR will bring is to challenge the current dogma of many disease mechanisms. In a sense, therapeutic editing is like the human equivalent of the knock out mouse. I predict that gene “therapeutic editing” will actually be diagnostic for specific disease mechanisms (or lack thereof). For instance some mutations that are high frequency “disease genes” are likely to be normal variants. Directly correcting these variants in patient derived cells would prove the lack of a disease. Editing will also be very valuable to determine if VUS mutations (variants of unknown significance) are really disease associated. Also it should be noted that there are many disease mutations that have controversial mechanism of action, that have not been solved since experiments in humans/human cells cannot be done. Now with the advent of iPSCs and CRISPR, function can be tested in human tissues. Eventually, somatic editing will be the ultimate test of the hypothesis, by editing directly in humans. Bruce Conklin, Gladstone Institutes, UCSF CRISPR has already tremendously impacted a large number of research areas. The most disruptive effects have been, in my personal experience, on animal genome engineering methods that used ES cells for homologous recombination and gene targeting that have been practiced for over three decades. In our lab, we have not handled any ES cells for 4 years! Channabasavaiah B. Gurumurthy, University of Nebraska Medical Center I think agriculture, and not therapeutics, will experience the earliest disruption. In fact, we are already witnessing the emergence of crops that have been “CRISPRed.” Examples include mushrooms that do not turn brown or tomatoes with improved traits. The technology may potentially help alleviate the problem of hunger or malnutrition in poorer regions of the world. This is a huge impact. Countries like China have gone into CRISPR-based genetic engineering of crops in a big way. Meng How Tan, Nanyang Technological University 4. What CRISPR applications do you think will be the first to reach the clinic? CRISPR/Cas9 system-based gene therapy for genetic disorders, such as muscular dystrophy, cystic fibrosis, and hemophilia. Kazuo Takayama, Osaka University I think gene therapy for some eye diseases, since the eye is an accessible and immunoprivileged organ. Meng How Tan, Nanyang Technological University Based on the ability to genetically modify cells outside of the patient’s body, targeting the hematopoietic system is likely to be one of the first approaches. In fact, some patients have already been treated for aggressive cancers by modifying cells of the immune system with CRISPR technology (Cyranoski, 2016). Jennifer A. Mitchell, University of Toronto For in vivo, anything that can be easily accessed, for example, skin diseases, eye diseases, and hearing loss. For ex vivo, hematopoietic diseases. Channabasavaiah B. Gurumurthy, University of Nebraska Medical Center 5. As this technology matures and becomes more accessible, how do you think it will affect natural ecosystems? I believe that agricultural use of CRISPR in crops or insects is the closest application of genome editing. However, uncertainties will be likely high in the use of multiplex editing via NHEJ. This is because these organisms are not subject to GMO regulation in many countries (based on the existence of transgenes or exogenous DNA), and also because there is currently no consensus in the assessment of off-target effects. Thus, the widespread and unregulated use of CRISPR in agriculture can potentially result in the release of modified organisms via NHEJ 301
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without sufficient environmental risk assessment. Tetsuya Ishii, Hokkaido University Bacteria and archaea evolved the CRISPR systems to protect their genomes from foreign nucleic acids; in that respect, CRISPR is already present in natural ecosystems. The ability to modify genomes is not new in biological science, but it has become easier with our recent understanding and use of the CRISPR system. As with any new technology, we need to proceed carefully and with regulatory oversight when considering how to apply it to human health and to other organisms outside of controlled laboratory settings. Jennifer A. Mitchell, University of Toronto It depends on how governments around the world regulate gene drives. There are two sides to the argumentgene drives can help control pests and harmful/undesirable organisms in the environment, but accidental or even malicious release of gene drives may result in irreversible damage to the natural ecosystem. Controlled release of genetically engineered organisms, like mosquitoes, is already happening in several parts of the world to limit the spread of infectious diseases, so the ecosystem will surely be affected in some way, but I hope the changes will mostly be in a good way. Meng How Tan, Nanyang Technological University 6. Which applications are you most excited to see CRISPR technologies applied to? While next-generation DNA sequencing has revolutionized our ability to “read” the genetic code, CRISPR technology is now enabling us to “write” and edit it. In addition to the therapeutic implications, CRISPR technology is also an incredible research tool to elucidate the function of genomes. In particular, CRISPRbased genetic screens (including loss-of-function CRISPRn and CRISPRi screens and gain-of-function CRISPRa screens) can reveal disease mechanisms and potential therapeutic targets. In combination with induced pluripotent stem cells from patients with disease mutations and CRISPR-corrected isogenic control cells, CRISPR-based genetic modifier screens can reveal the molecular mechanisms by which disease-associated mutations affect cellular function, and point to therapeutic strategies to correct cellular defects. Martin Kampmann, UCSF CRISPR has the power to bring “model organism genetics” to cancer drug development. Using CRISPR, we can make null alleles, point mutations, gene fusions, and other alterations, and we can do it in human cancer cell lines just as easily as we can do it in yeast or Drosophila. This vastly extends our ability to explore the architecture of cancer genomes and to probe these cells for therapeutic vulnerabilities. With CRISPR, we can delete a putative drug target and test whether the deletion phenocopies the effects of the drug. Or, we can introduce mutations throughout the target gene to identify alterations that confer drug resistance. If a gene is nonessential, then we can easily scan the genome for other mutations that confer synthetic lethality. By giving us a robust and flexible tool to edit the cancer genome, CRISPR will open new doors for the precise design and characterization of therapeutic molecules. Jason Sheltzer, Cold Spring Harbor Laboratory There are some very debilitating rare genetic diseases that affect children and are not inherited but are sporadic, meaning they are gene mutations that occur in the germ cells (sperm or ovum). One example is Hutchinson−Gilford progeria syndrome, which is usually first detected at 2 years of age and causes premature, rapid aging and death around 12 years of age. This disease cannot be treated with a gene therapy approach because the patient already has one copy of the normal protein. The problem is that the mutated gene produces a protein that functions
in a dominant way to override the normal function of the protein and cause premature aging. If we can improve the targeting efficiency of CRISPR genome editing approaches in patients so that we could correct the mutated gene in enough of the somatic cells of these children after they present with the disease at 2 years of age, we could vastly improve their quality of life and maybe even cure their disease. Jennifer A. Mitchell, University of Toronto While CRISPR has primarily been applied to gene knockouts, the technology offers effective means for broadly reshaping genomic structure. For research purposes, this includes the ability to knock-in reporter tags, thereby enabling one to monitor the specific molecular events underlying cellular physiology. To unravel the complexities of cell signaling, researchers have relied largely upon plasmid or viral expression systems for introducing fluorescent or luminescent reporters at expression levels typically far exceeding that of native genes. Unquestionably, this methodology has generated many high-profile discoveries. Yet, given the vital role of expression level on physiological function, the validity of this approach remains questionable. CRISPR avoids artifacts associated with ectopic expression by allowing direct integration of reporter tags into appropriate genetic loci. It also poses a new challenge in that native expression is 10 orders of magnitude below typical overexpression levels. Thus, reporter tags introduced using CRISPR should support very high detection sensitivity. By providing more accurate representation of native cellular physiology, along with the improving methodological efficiency of genome editing, the ability to incorporate reporter tags by CRISPR could change the way cell biologists approach their experiments. Marie Schwinn Thomas Machleidt, Kris Zimmerman, Christopher T. Eggers, Robin Hurst, Mary P. Hall, Lance P. Encell, Brock F. Binkowski, and Keith V. Wood, Promega Corporation; Andrew S. Dixon, University of Utah The discovery of CRISPR has provided scientists with a toolbox that has great potential to suppress and potentially eliminate mosquito vectors of human diseases, such as malaria, dengue, chikungunya, and Zika virus. Over 600 000 people die each year from malaria, most of whom are children under the age of five in sub-Saharan Africa, and over 50 000 000 people are infected with dengue each year, ∼10 000 of whom die from the disease. Given that all currently available tools are limited in their ability to control these diseases, we are excited by the potential of CRISPR-mediated gene drive technology to suppress mosquito vector populations and/or to render them incapable of transmitting diseases. These strategies have promise over a wide geographic scale and do not suffer from the human compliance issues that reduce the epidemiological impact of insecticidebased approaches and antimalarial drugs. Omar Akbari, UCSD and John Marshall, UC Berkeley We are most excited to see the application of CRISPR technologies in studies of epigenetic mechanisms during cell fate transitions, particularly to understand the precise temporal and spatial order of epigenetic modifications and the contribution of each chromatin modification to transcriptional regulation. Cas9 is already used effectively for targeted disruption of cis-regulatory DNA sequences, and dCas9 linked to various effector molecules is beginning to be utilized to interrogate the epigenome through targeted modifications. We envision further improvement of imaging tools that will allow us to read dynamic changes of histone and DNA modifications and chromatin conformation in living cells. These emerging CRISPR tools combined with fast developing high-throughput screening and single-cell sequencing 302
DOI: 10.1021/acschembio.8b00135 ACS Chem. Biol. 2018, 13, 296−304
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In Focus
research, we have capitalized on CRISPR-Cas9 to determine how molecular transport systems function in neurons. Molecular motors and their microtubule “highways” can be difficult to work with because of their size and stability, as well as number of subunits, isoforms, and binding partners. We are using CRISPRCas9 to replace genes with attP “docking sites” that facilitate the rapid and reliable knock-in of new alleles via site-specific recombination. We would love to visualize molecular motors and their cargos zipping along microtubules in neurons in intact animals, and we have had some success using fluorescent proteinbased approaches. But this an area that could really benefit from chemical biology: the development of bright, photostable fluorescent molecules that can be attached via small linkers to endogenous proteins and RNAs to enable the dynamic visualization of small numbers of molecules in animals. The intersection of chemical biology and CRISPR-Cas systems will undoubtedly continue to supply our toolbox with reagents to gain insights into how cells work. Jill Wildonger, University of Wisconsin Madison Chemical biologists can synthesize new, orthogonal small molecules that can be used to control the activity of CRISPR systems with high temporal precision and high spatial resolution in mammalian cells. Meng How Tan, Nanyang Technological University The optimization and modifications to the CRISPR system that have been developed to allow for programmable base editing, epigenome editing without changes to the DNA, and RNA editing are providing ever increasing tools to further our understanding of genome function. Chemical biology can contribute to the optimization of the efficiency and precise targeting of these tools, both requirements for their use in a clinical context. Jennifer A. Mitchell, University of Toronto 8. What are your major ethical concerns surrounding CRISPR applications? Unlike medical advances that people can choose to utilize or not as they see fit, ecological applications of CRISPR such as gene drive will necessarily affect everyone within an area. Developing these technologies according to the traditional closeted research model denies people a voice in decisions intended to affect them. Preregistering experiments and inviting concerns and suggestions at the earliest stages is more likely to lead to safer, more effective, and publicly supported applications. Yet, few scientists are willing to defy tradition and risk being scooped. Ensuring that all research proceeds in an ethical and trustworthy manner will require funders, journals, universities, and IP holders to change incentives in favor of open, preregistered CRISPR gene drive research. Kevin Esvelt, Massachusetts Institute of Technology The current recommendation from an American Society of Human Genetics (ASHG) workgroup, which included several international partners, is that it is inappropriate to perform germline gene editing that culminates in human pregnancy at this time (Ormond et al., 2017). Similarly, I do not support applying genome editing to the human germ line. It is important to ensure that regulatory systems are in place to establish appropriate guidelines and control the use of genome editing technology both in humans and in other organisms outside of controlled laboratory settings. Jennifer A. Mitchell, University of Toronto CRISPR has revolutionized biological research and is on a path to similarly change areas of broad societal import, from medicine to agriculture to the environment. In the medical arena, the potential to correct human mutations intersects with the growing availability of personal genomic information, raising far reaching questions about the future of human health interventions.
technologies have the potential to transform our understanding of the epigenetic mechanisms underlying cell fate transition during normal embryonic development, experimentally induced stem cell differentiation, and somatic cell reprogramming, as well as aberrant cell state changes in pathological conditions such as cancers. Huangf u Danwei and Julian Pulecio, Memorial Sloan Kettering Cancer Center As a card-carrying chemical biologist, I’m most excited to see how CRISPR can be interwoven to facilitate applications that have been core goals of our field. For example, chemical proteomics remains the gold standard for validation of drug-target occupancy and can also be used as a hypothesis-generating tool to explore how large enzyme families or protein chemotypes (such as reactive cysteines) vary with disease. However, one major challenge, particularly with exploratory profiling endeavors, is prioritizing functional hits for follow-up. Using chemical proteomic data to guide the design of focused CRISPRa and CRISPRi screens will enable our knowledge of physical protein− small molecule interactions (discovered using chemistry) to be translated into functional biology further, faster. The second area I’m excited to see genome-editing technologies make a difference (reflected in our submission for this special issue) is to study the mechanistic consequences of chromatin and RNA-modifying enzymes in living cells. The reason this is so important is because while modifications such as lysine acetylation are associated with active transcription, separating causation and correlation remains a major challenge. The ability to turn on specific enzymes as specific loci, as well as to perturb discrete domains within these enzymes using mutation or small molecules, should be clarifying. Finally, it is worth mentioning that one reason the CRISPR revolution is so captivating to chemical biologists is the rich history of small molecule sequence-specific nucleic acid targeting in our field, pioneered by Peter Dervan and others. It is interesting to speculate that, as CRISPR shows what applications are possible using programmable nucleic acid targeting (gene activation, repair, mutation, epigenome editing, et cetera), it may also fuel a renewed investment in the discovery of small molecule solutions to these problems. Jordan Meier, National Cancer Institute, National Institutes of Health I am tremendously excited by emerging applications of CRISPR. Although a lot of the spotlight has focused on therapeutic editing and disease models, there are many other applications that are equally exciting. For example, CRISPR editing of synthetic DNA arrays has been used for information storage in bacteria and lineage tracing in zebrafish. Also, CRISPR-based epigenetic modifiers can pinpoint functional elements in noncoding regions, modifying gene imprinting, and fine-tune gene expression. A remarkable property of CRISPR is that it can serve as a target platform for many other effector proteins. In computer science terms, it acts as a “pointer” to a specific location in the genome with limitless possibilities for what operations can be performed at these locations. Neville Sanjana, New York University 7. Are there specific areas of CRISPR research that could benefit from chemical biology? Yes, “targeted delivery”: developing new molecules that can escort CRISPR components to target tissues effectively and safely. Channabasavaiah B. Gurumurthy, University of Nebraska Medical Center Understanding how proteins and RNAs function in cells is fundamental to understanding how cells work. I am most excited by tools that enable us to manipulate and visualize molecules in intact cells in real time. The CRISPR-Cas systems have proven to be highly adaptable to the generation of such tools. In my own 303
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In Focus
In agriculture, the ability to edit plant and animal genomes without a trace challenges strongly held views on what is and is not a genetically modified organism and whether such organisms carry risks to human health or ecosystems. The potential of CRISPR-based gene drives opens questions about how we balance a desire to control disease vectors with concerns for unknown environmental impacts. As these examples make clear, CRISPR raises practical and ethical questions for individuals, societies, and humanity as a whole. We will best navigate the promises and challenges of the CRISPR revolution if the choices we make are deliberate and informed by diverse perspectives. It is therefore critical that everyonebiologists and nonbiologists alikegain an understanding of CRISPR’s capabilities, limitations, and risks. To that end, it is up to those of us with expertise in genome engineering to make the science of CRISPR and its applications broadly accessible. We can do this by engaging experts in relevant fields (ethics, history, and social policy to name a few) and the general public through our teaching (including courses for nonbiologists), outreach, writing, and interactions with the media. Kate O’Connor-Giles, University of WisconsinMadison I believe that most scientists are socially responsible, so my main concern is rogue agents laying their hands on gene editing technology to inflict harm on society. Meng How Tan, Nanyang Technological University 9. Could CRISPR have a bigger impact on our food supply? The growth of the global human population has led to a growing demand for food. Moreover, the average life expectancy continues to increase while global hunger is on the rise. According to “The State of Food Security and Nutrition in the World 2017,” more than 10% of the population is suffering from food insecurity. Undernutrition is a multidimensional problem, driven among other factors by climate change and political conflicts. Crop failures and the loss of livestock are jeopardizing the lives and livelihoods of millions of people. As the world faces famine conditions, we need to scale up our efforts to address this crisis. CRISPR not only holds great promise for human health but for our agriculture as well. One of the ethical concerns of CRISPR is if its applications will benefit all nations, and not just the industrialized ones. In other words, will the technology only be advantageous to people that are already advantaged? CRISPR offers a much needed short-cut to conventional breeding and could be harnessed to develop environmentally sustainable agriculture and food systems, so that people have access to a stable food supply. The technology could be exploited to generate more robust crops resistant to blights with increased yield and reduced fertilizer and water use. Ripening genes could be turned off in fruits and vegetables to slow down deterioration and reduce the losses often associated with the lack of refrigeration and inefficient transportation systems in developing countries. Achieving a world without hunger and malnutrition by 2030 is an ambitious goal set by the United Nations, and CRISPR should contribute to achieving this objective. Marie-Laurence Lemay and Sylvain Moineau, Université Laval
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