A Perspective on the State of Genome Editing
Authors: Dana Carroll
Journal: Molecular Therapy (2016)

Like the launch of a space probe, the advent of the CRISPR/Cas genome editing platform has caused both excitement and anxiety. The excitement, of course, reflects its high success rate, its simplicity, and its affordability.1Doudna JA Charpentier E Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 2014; 346: 1258096Crossref PubMed Scopus (3615) Google Scholar,2Hsu PD Lander ES Zhang F Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (3634) Google Scholar Its rapid adoption owes much to the groundwork laid by its predecessor nucleases, zinc-finger nucleases (ZFNs), meganucleases, and transcription activator–like effector nucleases (TALENs).3Carroll D Genome engineering with targetable nucleases.Annu Rev Biochem. 2014; 83: 409-439Crossref PubMed Scopus (382) Google Scholar All these reagents are able to make specific, targeted breaks in chromosomal DNA and generate both new mutations and intentional sequence changes. The anxiety arises from these same features of the technology and the prospect that some researchers might be too aggressive in using it.4Baltimore D Berg P Botchan M Carroll D Charo RA Church G et al.A prudent path forward for genomic engineering and germline gene modification.Science. 2015; 348: 36-38Crossref PubMed Scopus (432) Google Scholar Moreover, if it is really true that CRISPR/Cas genome modifications can be accomplished in anyone's garage, will it be employed for nefarious purposes? The International Summit on Human Gene Editing, hosted by the scientific academies of China, the United Kingdom, and the United States in Washington, DC, in December 2015, was called to address the societal issues surrounding such genome modifications, in the context of what the technology can currently accomplish. The particular focus was the potential for editing the human germ line, a prospect that is gathering increasing attention. The summit concluded with a statement that it would be irresponsible to pursue germline editing currently, although research in this direction is warranted while broad discussions of the context and consequences continue.5Baltimore D Baylis F Berg P Daley GQ Doudna JA Lander ES et al.On human gene editing: International Summit Statement. 3 December 2015, 2015Google Scholar Another recommendation of the summit was that somatic therapies based on genome editing should proceed, within the existing regulatory and clinical systems. Despite all the optimistic talk in this realm, very few clinical uses have actually been undertaken. We are aware of the phase I trial using ZFNs ex vivo to knock out CCR5 in T cells of patients infected with HIV-1, with the aim of creating cells resistant to killing by the virus.6Tebas P Stein D Tang WW Frank I Wang SQ Lee G et al.Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.N Engl J Med. 2014; 370: 901-910Crossref PubMed Scopus (1011) Google Scholar At the Washington summit, Fyodor Urnov of Sangamo Biosciences reported that more than 80 people have now been treated safely in this protocol. An extension of this program would make the modification in hematopoietic stem cells from the bone marrow so as to provide long-lasting protection, and this is being evaluated in an open clinical trial. Urnov was also able to report US Food and Drug Administration clearance of an exciting new trial: in vivo treatment of hemophilia B using ZFNs. This application breaks new ground in three respects.7Sharma R Anguela XM Doyon Y Wechsler T DeKelver RC Sproul S et al.In vivo genome editing of the albumin locus as a platform for protein replacement therapy.Blood. 2015; 126: 1777-1784Crossref PubMed Scopus (213) Google Scholar First, it is an in vivo treatment, with the therapeutic materials delivered to the intact liver, rather than treatment of cells ex vivo. Second, the therapeutic coding sequence will be inserted at a “safe harbor” site, rather than its natural genomic location. Third, it requires the introduction of a donor DNA that carries the corrected gene sequence. Typically the efficiency of homologous repair with such a donor is substantially lower than simple mutagenesis at the nuclease target, but in this disease, even a low level of expression of factor IX would be beneficial. Somatic therapies will continue to be developed. In vivo editing approaches will require additional developments in the areas of efficacy and, in particular, delivery of the reagents. As additional ex vivo stem cell approaches are perfected, genome editing is an obvious adjunct, particularly for autologous transplants. Personal ex vivo treatments are too expensive to be used very broadly. One solution is to delete cell surface components to prevent both graft-vs.-host and host-vs.-graft reactions.8Torikai H Reik A Soldner F Warren EH Yuen C Zhou Y et al.Toward eliminating HLA class I expression to generate universal cells from allogeneic donors.Blood. 2013; 122: 1341-1349Crossref PubMed Scopus (202) Google Scholar In one example, off-the-shelf CAR T cells of this type have been created using TALENs and delivered to a very young leukemia patient in Britain.9Frederickson RM A new era of innovation for CAR T-cell therapy.Mol Ther. 2015; 23: 1795-1796Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar While ZFN and TALEN products have made it to the clinic, CRISPR/Cas is making important contributions in discovery research and human disease modeling. Again following precedents from the earlier platforms, CRISPR/Cas tools have been used to create and to correct analogs of human disease mutations in the genomes of mice,10Wang H Yang H Shivalila CS Dawlaty MM Cheng AW Zhang F et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell. 2013; 153: 910-918Abstract Full Text Full Text PDF PubMed Scopus (2594) Google Scholar rats,11Ma Y Shen B Zhang X Lu Y Chen W Ma J et al.Heritable multiplex genetic engineering in rats using CRISPR/Cas9.PLoS One. 2014; 9: e89413Crossref PubMed Scopus (79) Google Scholar pigs,12Li F Cowley DO Banner D Holle E Zhang L Su L Efficient genetic manipulation of the NOD-Rag1–/–IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology.Sci Rep. 2014; 4: 5290PubMed Google Scholar monkeys,13Chen Y Zheng Y Kang Y Yang W Niu Y Guo X et al.Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9.Hum Mol Genet. 2015; 24: 3764-3774Crossref PubMed Scopus (177) Google Scholar and other organisms. Because this has been accomplished so readily and so broadly in mammals via embryo injection, it makes analogous modifications of the human germ line seem very close at hand. In fact, one published study describes Cas9-mediated editing in human embryos that were inherently unviable and never capable of initiating a pregnancy.14Liang P Xu Y Zhang X Ding C Huang R Zhang Z et al.CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.Protein Cell. 2015; 6: 363-372Crossref PubMed Scopus (760) Google Scholar The conclusions of the Washington summit, and much of the ethical argument that human germline editing should not be pursued currently, arise from the prevailing opinion that the efficiency, the accuracy, and the specificity of the process are not sufficiently robust to ensure efficacy and safety. Although true, this view is rather short-sighted. I think we should acknowledge that human germline editing is going to happen. In addition, progress will be made toward solving the technology-based efficacy and safety issues. In that case, what milestones need to be met for the biomedical community to be comfortable with offering it as a potential therapy? On the technological side, we still have a long way to go. We need assurance that off-target cleavage will not lead to mutations that will compromise expected benefits. Good progress is being made in both minimizing15Fu Y Sander JD Reyon D Cascio VM Joung JK Improving CRISPR-Cas nuclease specificity using a modified guide RNA architecture.Nat Biotechnol. 2014; 32: 279-284Crossref PubMed Scopus (1391) Google Scholar,16Mali P Aach J Stranges PB Esvelt KM Moosburner M Kosuri S et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nat Biotechnol. 2013; 31: 833-838Crossref PubMed Scopus (1314) Google Scholar,17Ran FA Hsu PD Lin CY Gootenberg JS Konermann S Trevino AE et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell. 2013; 154: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (2346) Google Scholar,18Slaymaker IM Gao L Zetsche B Scott DA Yan WX Zhang F Rationally engineered Cas9 nucleases with improved specificity.Science. 2016; 351: 84-88Crossref PubMed Scopus (1526) Google Scholar and detecting19Koo T Lee J Kim J-S Measuring and reducing off-target activities of programmable nucleases including CRISPR-Cas9.Mol Cells. 2015; 38: 475-481Crossref PubMed Scopus (145) Google Scholar off-target effects, but the bar should be set very high before heritable modifications are pursued. Currently we have inadequate control over the types of modifications produced at the genomic target. Following CRISPR/Cas (or ZFN or TALEN) cleavage, cellular repair processes can either introduce local mutations or copy sequences from a homologous donor DNA. For most therapeutic applications, the latter process is desirable, but the former typically dominates and must be minimized. A great deal of current research is directed toward addressing this issue, but no generally effective approach has yet emerged. On the clinical side, we need to identify conditions that are reasonable candidates for germline treatment. It has been pointed out that there are effective alternatives in current practice, particularly preimplantation screening.20Lanphier E Urnov F Haecker SE Werner M Smolenski J Don't edit the human germ line.Nature. 2015; 519: 410-411Crossref PubMed Scopus (336) Google Scholar These alternatives have their own drawbacks, both practical and ethical. It seems likely that germline gene correction may ultimately be offered as an alternative to such procedures, once technical standards have been met. Perhaps most important, as emphasized by a number of presentations at the Washington summit, we must look very deeply into the societal aspects of employing this powerful technology. It will not be possible to allay the concerns of all groups––it never is––and some will resist the fundamental appearance that we are “playing God.”21Carroll D Carroll D The societal opportunities and challenges of genome editing.Genome Biol. 2015; 16: 242Crossref PubMed Scopus (52) Google Scholar On the other side will be patient advocates and disease-specific organizations that will promote any therapy that improves the prospects for potential sufferers of devastating conditions, and physicians will want to offer all effective treatments. Other societal issues that deserve thorough discussion are the following. Will germline gene editing always be a treatment reserved for the rich and well connected? Are there ways to distribute these therapies more broadly, and can approaches be developed specifically for the disadvantaged? What conditions will be treated with the technology? Can we avoid reinforcing stereotypes by choosing what constitutes a condition in need of treatment? One instructive example is hereditary deafness: is it a disease? How can we prevent narcissistic, cosmetic, and malevolent uses of the technology? These are thorny issues with no analytical solutions. Quite sensibly, the Washington summit began to address all of these challenges, and its final statement called both for research that would begin to resolve the technical issues and for much broader and more extensive discussions. In the wake of the summit, the National Academies of Sciences and Medicine established a study committee to continue examination of the impact of the technology.22The National Academies of Sciences, Engineering, and Medicine Academies' Consensus Study on Human Gene Editing Begins: First Data-Gathering Meeting Feb. 11–12,. 8. December 2015, 2016Google Scholar The prospects for applying genome editing in clinical therapies are enticing. In practice, there is much to be done en route. The views expressed in this Commentary are those of the author and do not necessarily reflect the official position of the Journal and its editorial team, or that of the American Society of Gene and Cell Therapy and its officers.