CRISPR Ethics: Moral ConsiderationsforApplicationsofa .

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ReviewKDCYJMBI-65762; No. of pages: 14; 4C:CRISPR Ethics: MoralConsiderations for Applications of aPowerful ToolCarolyn Brokowski 1 and Mazhar Adli 21 - Department of Emergency Medicine, Yale School of Medicine, 464 Congress Avenue, New Haven, CT 06519-1362, USA2 - Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue,Charlottesville, VA 22908, USACorrespondence to Mazhar Adli: 8.05.044Edited by Prashant MaliAbstractWith the emergence of CRISPR technology, targeted editing of a wide variety of genomes is no longer anabstract hypothetical, but occurs regularly. As application areas of CRISPR are exceeding beyond researchand biomedical therapies, new and existing ethical concerns abound throughout the global community aboutthe appropriate scope of the systems' use. Here we review fundamental ethical issues including the following:1) the extent to which CRISPR use should be permitted; 2) access to CRISPR applications; 3) whether aregulatory framework(s) for clinical research involving human subjects might accommodate all types of humangenome editing, including editing of the germline; and 4) whether international regulations governinginappropriate CRISPR utilization should be crafted and publicized. We conclude that moral decision makingshould evolve as the science of genomic engineering advances and hold that it would be reasonable fornational and supranational legislatures to consider evidence-based regulation of certain CRISPR applicationsfor the betterment of human health and progress. 2018 Elsevier Ltd. All rights reserved.IntroductionThe CRISPR (Clustered Regularly InterspacedShort Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) system (“CRISPR” or “the system”) isthe most versatile genomic engineering tool createdin the history of molecular biology to date. Thissystem's ability to edit diverse genome types withunprecedented ease has caused considerable excitement and alarm throughout the internationalbiomedical community.CRISPR appears to offer considerable promise ina wide variety of disease contexts. For example,around the world at least 15 clinical trials—focusedon multiple myeloma; esophageal, lung, prostate,and bladder cancer; solid tumors; melanoma;leukemia; human papilloma virus; HIV-1; gastrointestinal infection; β-thalassemia; sickle-cell anemia;and other diseases—involving CRISPR applicationshave been developed [1–3]. Moreover, as of May,2018, in China at least 86 individuals have had theirgenes altered as part of clinical trials [4].0022-2836/ 2018 Elsevier Ltd. All rights reserved.While significant public support exists for therapeutic applications [5], ethical (moral) and safetyconcerns about certain areas of CRISPR applications, such as germline editing, are apparent aroundthe world [6]. Notably, such discussions commencedduring the Napa Valley meeting of 2015 when aleading group of CRISPR–Cas9 developers, scientists, and ethicists met to examine the biomedical,legal, and ethical aspects of CRISPR systems [7].From this meeting, more extensive deliberationswere solicited, and the United States (US) NationalAcademies of Sciences, Engineering, and Medicine(NASEM or “The Committee”) invited the ChineseAcademy of Sciences and the United Kingdom's(UK) Royal Society to participate in the InternationalSummit on Human Gene Editing [8]. The goal of thismeeting was to examine when, where, and how thetechnology might be applied in humans. Thisdiscussion continued in February of 2017 when amultidisciplinary committee of the NASEM publisheda comprehensive report examining numerous aspects of human genome editing [9].J Mol Biol (2018) xx, xxx–xxxPlease cite this article as: C. Brokowski, M. Adli, CRISPR Ethics: Moral Considerations for Applications of a Powerful Tool, J. Mol. Biol.(2018), https://doi.org/10.1016/j.jmb.2018.05.044

2Review: CRISPR EthicsTo date, the NASEM report provides perhaps themost influential, extensive analysis examining wideranging concerns about human genome editing [10].Importantly, the Committee favored somatic genomeediting, but did not permit genomic modification forany kind of enhancement [9, 11]. Also, thoughimpermissible at present, the Committee concludedcautiously that human heritable genome editing, themodification of the germline with the goal of creatinga new person who could potentially transfer thegenomic edit to future generations, would bepermissible under certain conditions: “In light of thetechnical and social concerns involved heritablegenome-editing research trials might be permitted,but only following much more research aimed atmeeting existing risk/benefit standards for authorizing clinical trials and even then, only for compellingreasons and under strict oversight.” [9] Although bylaw, US federal funding cannot be used to supportresearch involving human embryos [12–14], theNASEM report suggests that when technical andsafety risks are better understood then clinical trialsinvolving germline editing might begin [9].In this review, we aim to summarize fundamentalethical concerns about CRISPR use in general, butthe list is not exhaustive. First, we briefly reviewCRISPR systems and their applications in editinggenomes and epigenomes. Second, we describehow complexities of CRISPR science affect those ofCRISPR ethics and vice versa. Third, we assessseveral key ethical considerations. Notably, whilesome of these concerns are specific to CRISPRtechnology, many, such as research on humanembryos, have been debated long before theCRISPR revolution [15]. Moreover, since CRISPRis still a maturing technology, novel applications inthe future may raise new ethical quandaries meritingfurther attention and dissection. Fourth, it is important to point out that, though morality and law oftenoverlap, significant differences exist. Although lawmay affect ethics and vice versa, we focus mostly onethics. Finally, while discussing these issues, weassume no position on any topic; our account ismerely descriptive. Therefore, we make no attemptto settle any of the controversies presented herein.CRISPR systems and their usesDifferent CRISPR systems in genome editingCRISPR is a natural bacterial defense systemagainst invading viruses and nucleic acids. Overbillions of years, multiple CRISPR-type immunesystems have evolved. Naturally occurring CRISPRsystems are typically classified by their repertoires ofCRISPR-associated (cas) genes, which are oftenfound adjacent to the CRISPR arrays [16, 17].Although the characterization is yet to be finalized,two major classes of CRISPR–Cas adaptive immunesystems have been identified in prokaryotes [18–20].This division is based on the organization of effectormodules. Class 1 CRISPR–Cas systems employmulti-protein effector complexes and encompassthree types (I, III, and IV). By contrast, Class 2systems utilize single protein effectors and encompass three other types (II, V, and VI). Although variousnatural CRISPR–Cas systems have been repurposedfor genome editing, due to its robust gene-editingefficiency and broader genome-targeting scope owingto its simple NGG PAM sequence requirement, theCas9 from Streptococcus pyogenes (spCas9) iscurrently the most commonly used CRISPR–Cas9protein. It is worth noting that multiple efforts areunderway to discover novel Cas9 variants or reengineer the existing Cas9 proteins, which will haveless dependence on the stringent PAM-sequencerequirement [21, 22].CRISPR goes beyond genome editingThe DNA-editing capacity of CRISPR–Cas9 is dueto the ability of the WT Cas9 protein to cause doublestranded breaks at the target site that is determinedby the custom-designed short guiding RNA [23]. Therepair of DNA breaks frequently results in indels, dueto the non-homologous end joining (NHEJ) repairmechanism. However, when a complementary template is available, homology-directed repair (HDR)machinery can use it and thereby achieve moreprecise gene editing. Notably, a single-point mutationin either of the two catalytic domains of Cas9 resultsin a nickase Cas9 (nCas9), whereas mutations inboth domains (D10A and H840A for spCas9)diminish Cas9's catalytic activity, resulting in deadCas9 (dCas9) [24]. Interestingly, the applicationareas of modified Cas9 proteins are exceeding thatof WT Cas9 [25]. Such uses are largely possiblebecause the nCas9 or dCas9 can still be guided tothe target sequence [26]. Researchers employedthese Cas9 variants for unique purposes. Forexample, tandem targeting of nCas9 has beenutilized to improve targeting specificity [27, 28].More recently, this enzyme has been used as thebase platform for second generation genome-editingtools called “base editors” [29, 30] Base-editingtechnology employs cytidine or adenine deaminaseenzymes to achieve the programmable conversion ofone base into another (C to T or A to G). Mostimportantly, the targeted base transition happenswithout DNA double-stranded breaks [29, 31]. Werecently utilized this technology to edit the universalgenetic code and introduced a “stop” codon in thegenes [32].In addition to nCas9, researchers utilized theguidable capacity of dCas9 as a platform to recruitvarious effector proteins to a specific locus in livingcells. Generally, these activities can be classified asPlease cite this article as: C. Brokowski, M. Adli, CRISPR Ethics: Moral Considerations for Applications of a Powerful Tool, J. Mol. Biol.(2018), https://doi.org/10.1016/j.jmb.2018.05.044

3Review: CRISPR Ethicsepigenetic editing (to alter locus-specific epigeneticinformation), gene regulation (to turn the activities ofsingle or multiple genes on or off), chromatin imaging(to label and monitor chromatin dynamics in livingcells), and manipulation of chromatin topology (toalter 3D chromatin structure in the nuclear space)[33].CRISPR research is progressing at a rapid pace.Recently, scientists have also uncovered newCRISPR–Cas systems (Cas13) that can target RNAinstead of DNA [34, 35]. By enabling targeted RNArecognition and editing, these newer RNA-targetingCRISPR tools have their unique applications rangingfrom biomedical and biotechnological to the detectionof nucleic acids [36, 37]. Although many ethicalconcerns are related to the catalytic activities of WTCas9—partly because it permanently alters thegenetic information—some of these activities ofcatalytically inactive dCas9, nickase-Cas9-basedplatforms, such as base editors and recently discovered RNA-targeting Cas proteins, may raise comparable moral issues depending on the duration of theexerted effect and the purpose of the experiments.Detailed discussion of such issues, however, isbeyond the scope of this review (Table 1).CRISPR ethics and science:Uncomfortable bedfellowsMoral decisions, especially in biomedicine, areempirically informed and involve assessing potentialrisk-benefit ratios—attempting to maximize the latterwhile minimizing the former. To navigate ethicaldecision making, it is critical to consider the range ofpossible outcomes, the probabilities of each instantiating, and the possible justifications driving the results ofany one. The ethical concerns about CRISPR genomeengineering technology are largely due to at least threeimportant reasons.First, there are concerns about the power andtechnical limitations of CRISPR technology. Theseinclude the possibilities of limited on-target editingefficiency [38, 39], incomplete editing (mosaicism)[40, 41], and inaccurate on- or off-target editing [42,43]. These limitations have been reported in CRISPRexperiments involving animals and human cell lines.However, the technology is evolving at an unprecedented pace. As more efficient and sensitiveCRISPR tools are developed, many of these concerns may become obsolete. Yet for the sake of thisreview, we mention these limitations as one of theprincipal worries about widespread CRISPR utilization. Second, it is unclear whether modified organisms will be affected indefinitely and whether theedited genes will be transferred to future generations,potentially affecting them in unexpected ways.Combined with technical limitations and the complexities of biological systems, making precisepredictions about the future of an edited organismand gauging potential risks and benefits might bedifficult, if not impossible. Thus, uncertainty resultingfrom these factors hinders accurate risk-benefitanalysis, complicating moral decision making.Finally, the skeptical view is that even if the genomeis edited as expected and the desired functional outputis achieved at the given time, the complex relationshipbetween genetic information and biological phenotypesis not fully understood. Therefore, the biologicalconsequence of editing a gene in germline and/orsomatic cells may be unclear and unpredictabledepending on the context. Many biological traits aredetermined by the complex regulatory actions ofnumerous genes. Hence, is it difficult, if not impossible,to “design” a biological phenotype at the wholeorganism level. Across biological outcomes, whetherin normal or in disease development, it is uncommonthat a single gene is the only factor shaping a complexbiological trait. Other genetic regulatory factors such asadditional genes or distal regulatory elements (e.g.,enhancer or repressor elements), as well as environmental and epigenetic factors, contribute to theemergence of a biological phenotype. To argue thatmodifying a gene changes a desired phenotype (undercertain conditions) implies at least a reasonableunderstanding of other independent variables contributing to the phenotype's instantiation. But this understanding is still far from complete in many normal anddisease processes [44, 45]. Given the uncertaintyregarding how gene expression and modificationinfluence complex biological outcomes, it is difficult toappraise potential risk and benefit. This ambiguitycreates a challenge on its own and is one of the sourcesobscuring efficient ethical deliberation and decisionmaking.Nevertheless, regulations governing cellular- andgene-therapy research may facilitate the safe development and oversight of some clinical trials involvingCRISPR-based-editing applications. In the UnitedStates, for instance, cellular- and gene-therapyproducts, including many CRISPR applications, atthis time are defined generally by the Food and DrugAdministration as biological products and are regulated by the Food and Drug Administration's Centerfor Biologics Evaluation and Research/Office ofCellular, Tissue, and Gene Therapies [46–48].Although the risks and benefits of many suchtherapies increasingly are better understood [49],questions regarding safety and efficacy remain.Thus, future advancements likely will continue toimprove the benefits of this revolutionary technology,while minimizing the potential risks. Regardless ofthe uncertainty posed by novel CRISPR technologiesand applications, in several locations around theworld significant regulatory frameworks exist bywhich risks may be monitored and contained.However, wherever such infrastructure and oversightare lacking, safety and privacy risks might increase.Please cite this article as: C. Brokowski, M. Adli, CRISPR Ethics: Moral Considerations for Applications of a Powerful Tool, J. Mol. Biol.(2018), https://doi.org/10.1016/j.jmb.2018.05.044

4Review: CRISPR EthicsTable 1. Risk-benefit considerations in CRISPR technologyBenefit(s)Risk(s)/Harm(s)Basic and pre-clinical research New model organisms and cell linesIncreased gene-editing efficiencyHigh-throughput screensNovel drug targetsAccess to totipotent cellsIdentification of novel signaling,regulatory, and developmental pathways Development of novel gene-editingapproaches (base editing and RNA targeting) Knowledge advancement Experimentation involving human embryos iscontroversial and illegal in some countries Potential for privacy and confidentiality breachesTranslational andclinical medicine Serious injury, disability, and/or death to researchparticipant(s) and/or offspring Blurry distinction between therapeutic andenhancement applications, leading to potentialsubtle or obvious exacerbation of inequalities Misapplications Eugenics Potential for inequitable access and exacerbationof inequalities ImmunotherapyOrganoidsNovel drug targetsArtificial intelligenceModification of pathological genesNovel therapeutics and fertilityapplicationsProcreative libertyAbility to “fix” single base changesKnowledge advancementPotential for equitable accessNon-therapeuticapplications Enhancement to augment select faultyor normal human characteristics Fortification of crops and livestock Successful control of pests, invasivespecies, and reservoirs (gene drives) Disease/infection control (e.g., malaria,dengue fever, Lyme and Chagasdisease, schistosomiasis) Ecosystem alteration to protectendangered species (gene drives) Safety Crop cultivation Knowledge advancement Access to CRISPRtechnology Price gouging Prohibitively expensive applicationsRegulations forclinical research involvinghuman subjects Established framework in somecountries to manage research risks Legal mechanisms for redressalready exist, depending on location Lack of appropriate supervisory infrastructure,oversight, and/or regulatory frameworkin many nations Unclear how to supervise the research even insome countries with regulatory oversight Over-regulation might hinder progressNational and internationalregulations, law, and policy Prevention against misuses of technology Safeguard against risky, potentiallyharmful conditions Potential to encroach on individual, scientific,and societal autonomy Limit discovery and progress Difficult enforcement Lack of uniformity may create inconsistenciesin applications of laws/regulationsInexpensive (technology itself)Widely availableProfit, economic growthInnovationEthical concernsTo what extent should CRISPR experimentationbe permitted in basic and pre-clinical biomedicalresearch?Although it is less than a decade old, CRISPR–Cas9 has demonstrated unprecedented potential toEugenicsExacerbation of racism and inequalityTheoretical risk for damage to ecosystemsTheoretical risk of misuserevolutionize innovation in basic science. From virusesand bacteria [50, 51], to simple model organisms, suchas Drosophila melanogaster (fruit fly) [52], Anophelesgambiae (mosquito) [53], Saccharomyces cerevisiae(budding yeast) [54], Hydra magnipapillata (hydra)[55], Caenorhabditis elegans (round worm) [56],Danio rerio (zebra fish) [57], and Arabidopsis thaliana(rockcress) [58], to larger animals such as pigs [59],cattle [60], and monkeys [61], and even humanPlease cite this article as: C. Brokowski, M. Adli, CRISPR Ethics: Moral Considerations for Applications of a Powerful Tool, J. Mol. Biol.(2018), https://doi.org/10.1016/j.jmb.2018.05.044

5Review: CRISPR Ethicszygotes [62, 63], CRISPR experimentation has led tonovel, important findings. Such benefits include at leastthe following: increased overall efficiency in geneediting compared with previous genomic engineeringtechniques like transcription activator-like effectornucleases (TALENs) and zinc finger nucleases(ZNFs) [64]; significant insights into the evolutionarytransformation of fish fins into tetrapod limbs [65];investigation into new organisms [66]; genetic andepigenetic screens [67, 68]; the creation of novel celllines [69]; high-throughput screens and libraries [70];the elucidation of novel genomic and epigenomicregulatory pathways [71, 72]; insights into the development of butterfly coloring and patterning [73]; thefunctional characterization of key genes and molecularsignaling pathways [74, 75]; and drug-targetingscreens [76, 77]. Data from such experimentationprovide essential clues and understanding that promote biomedical discovery, advancement, and thebasis for potential medical benefits.One of the major controversies about CRISPRtechnology emerges from its possible applicat

CRISPR ethics and vice versa. Third, we assess several key ethical considerations. Notably, while some of these concerns are specific to CRISPR technology, many, such as research on human embryos, have been debated long before the CRISPR revolution [15]. Moreover, since CRISPR is still a maturing technology, novel applications in

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