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Opinion
Germline genome-editing research and its socioethical implications Tetsuya Ishii Office of Health and Safety, Hokkaido University, Sapporo 060-0808, Hokkaido, Japan
Genetically modifying eggs, sperm, and zygotes (‘germline’ modification) can impact on the entire body of the resulting individual and on subsequent generations. With the advent of genome-editing technology, human germline gene modification is no longer theoretical. Owing to increasing concerns about human germline gene modification, a voluntary moratorium on human genome-editing research and/or the clinical application of human germline genome editing has recently been called for. However, whether such research should be suspended or encouraged warrants careful consideration. The present article reviews recent research on mammalian germline genome editing, discusses the importance of public dialogue on the socioethical implications of human germline genome-editing research, and considers the relevant guidelines and legislation in different countries. Urgent calls for a moratorium on human germline genome editing Rapid technological advances in the past decade have enabled mammalian germline gene modification. Notably, injecting a self-inactivating lentiviral vector into non-human primate embryos resulted in the generation of transgenic monkeys from which germline transmission was attained [1]. However, this methodology requires viral vectors which are frequently difficult to handle in the laboratory, and is largely limited to gene addition. More recently, genome editing, including the use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/ Cas (CRISPR-associated) systems such as Cas9, has demonstrated efficient genetic modification in non-human primates [2,3] as well as in human embryonic stem cells (hESCs) (reviewed in [4]). Although mammalian cells have been frequently been transduced by either integrating or non-integrating viral vectors harboring genome-editing nucleases, customized nucleases can, in some cases, be introduced into cells as an RNA or protein, together with a DNA template (if needed). More importantly, these programmable nucleases, particularly Cas9, are much easier to use for various types of gene Corresponding author: Ishii, T. ([email protected]). Keywords: germline genome editing; CRISPR/Cas9; ethics; genome engineering; embryonic stem cells. 1471-4914/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2015.05.006
modification, and thus are now being used instead of conventional genetic engineering in many laboratories worldwide. The robustness of this genome engineering technology has made it conceivable that gene modification of the human germline (oocytes, sperm, zygotes, and embryos) (Box 1) is becoming feasible in the clinical setting [4]. However, this type of gene modification has raised tremendous debate in the context of medical beneficence, safety concerns, challenges to human dignity, and risk of abuse for eugenics or enhancement (the parental pursuit of specific traits for non-medical reasons) [5]. Consequently, many countries forbid human germline gene modification for reproductive purposes [4,6]. Recently, representatives of the Alliance for Regenerative Medicine, a group of interested stakeholders including Cas9 developers and the International Society for Stem Cell Research (ISSCR), have called for a voluntary moratorium on research into and/or the clinical application of human germline genome editing owing to increasing bioethical concern [7–9]. These calls could be welcome because a moratorium might provide opportunities to reach a domestic or international consensus regarding whether society accepts or rejects human germline gene modification for medical purposes. The first call asked researchers not to carry out either research in the field or clinical application of the findings [7]. The latter two calls requested a moratorium on the clinical application of genome engineering, but suggested that in vitro research on editing the human germline genome should be permitted [8,9]. However, as demonstrated by the history of assisted reproductive technology (ART), the outcomes of human embryo research send a strong message to the public [10]. Meanwhile, draft regulations allowing mitochondrial donation, which changes the mitochondrial DNA content by nuclear transfer between oocytes or embryos to prevent maternal transmission of serious mitochondrial diseases, have recently been approved in the UK (effective on October 29, 2015) [11]. Although a controversial germline gene modification that has garnered intense attention worldwide – from initial consideration by the Human Fertilisation and Embryology Authority (HFEA) to the vote in Parliament – the UK became the first nation to legalize a form of human germline gene modification, changing the international regulatory landscape [6]. This opinion article reviews recent developments in mammalian germline genome-editing research. In addition, the significance of a public dialogue regarding the socioethical implications of Trends in Molecular Medicine xx (2015) 1–9
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Opinion Box 1. Human germline gene modification and clinical cases In a sexually reproducing organism, the genetic information for an individual is derived from oocytes and spermatozoa. Genetically modifying germ cells or zygotes (one-cell-stage embryos) can impact on the entire body of the offspring, and on subsequent generations via modified germ cells. Therefore, germline gene modification has been considered to be efficacious against genetic diseases. Two forms of germline gene modification focused on mitochondria (that contain their own genome) have been performed for treating infertility in the clinical setting. Case 1. Ooplasmic transfer Ooplasmic transfer is technically based on intracytoplasmic sperm injection (ICSI) for treating male infertility [13]. A small portion of the cytoplasm from donor oocytes (ooplasm) is transferred, together with a spermatozoon, into quality-compromised oocytes to restore their developmental potential. The mitochondria in the ooplasm were considered to be one of the key factors involved in the improved fertility in animal studies [59,60]. Cohen et al. reported the first case of pregnancy and childbirth in 1997 [61]. With the consent of the donors, this technique was applied in the USA. Heteroplasmy was confirmed in the blood of two infants born via this technique [62]. However, two congenital anomalies, 45 XO (Turner syndrome) and pervasive developmental disorder (PDD), were found in other children [63]. Subsequently, the FDA decided to regulate this treatment owing to the potential health risk to offspring. Meanwhile, ooplasmic transfer had already assisted nearly 30 childbirths worldwide [62]. In 2014, Alana Saarinen was introduced as one of the offspring (http://www.bbc.com/news/ magazine-28986843). She appeared to be healthy. Case 2. Pronuclear transfer Pronuclear transfer aims to exchange the pronuclei between two different one-cell-stage embryos. In 2003, Zhang et al. reported the first case of a pregnancy following pronuclear transfer carried out in China [64]. The transfer of the five treated embryos resulted in a triplet pregnancy with fetal heartbeats. A fetus was reduced for better development of the other two. However, these fetuses died of respiratory distress and cord prolapse, respectively. The mitochondrial DNA profiles of the fetal cells were similar to those from the donor. It was concluded that pregnancy with a normal karyotype is attainable by pronuclear transfer. However, the use of this procedure resulted in the establishment of the Guidelines on Human Assisted Reproductive Technologies (2003), which forbid using ooplasmic or nuclear transfer as well as genetic modification of the germline for reproductive purposes [4].
in vitro research on editing the human germline genome is discussed, while reviewing the guidelines and legislation that govern human embryo research, as well as reproduction following germline gene modification, in different countries across the world. Genome editing-mediated germline gene modification Genome editing can directly modify a gene within the genome, while conventional genetic engineering involves introducing an additional gene copy (reviewed in [12]). Introducing bacterial nucleases customized to home to a specific sequence into cells or embryos initiates a desired genetic change by creating double-strand breaks (DSBs) in the target sequence. Subsequently, the DSBs are repaired via non-homologous end-joining (NHEJ) in the absence of a repair template, or by homology-directed repair (HDR) if provided with a template. The NHEJ pathway efficiently results in a small insertion or deletion (termed ‘indel’) at a target site, whereas HDR can repair a mutation or add a desired DNA sequence at a specific site. 2
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For germline gene modification, the programmable nucleases (together with single guide RNAs in the case of Cas9) are directly injected into animal one-cell-stage embryos (reviewed in [4]). Embryo microinjection – which is outwardly similar to a common ART technique, intracytoplasmic sperm injection (ICSI) [13] – demonstrated efficient generation of genetically modified animals including mice [14–29], rats [30–32], pigs [33–35], sheep [36], cattle [33,36], and non-human primates [2,3,37], via NHEJ or the HDR pathway (Table 1). Moreover, germline gene modifications are also attainable via genome editing of spermatogonial stem cells (SSCs), potentially preventing mosaicism in offspring by avoiding the totipotent states that accompany embryogenesis. Successful editing using the SSC approach has been demonstrated in mice [38] and in rats [39]. Of note, Reddy et al. demonstrated that mitochondria-targeted TALENs (mito-TALENs) can be used in mouse oocytes fused with cells from a patient to eliminate human mitochondrial DNA (mtDNA) mutations [40]. More recently, Cas9-mediated gene modification was reported using human tri-pronuclear zygotes [41] (Table 1). However, the efficiency of HDR of the b-globin gene (HBB) was 4.7% (per injected zygote) and the modified embryos displayed mosaicism in which wild type cells and genetically modified cells coexisted. This latter might be due to the use of abnormally fertilized embryos. In addition, Cas9 treatment apparently induced off-target mutations. These results suggest that there is room to optimize the protocol for germline genome editing in terms of (i) the methodology used to introduce the nucleases (the dose and the form of the enzymes – plasmid, mRNA, or protein [42,43]; cytoplasmic or pronuclear injection), (ii) mosaicism [14,19,20,24,25,28,30–32,34,37], and (iii) possible off-target mutations [21–24,27,30,31] (Table 1). The risk of offtarget mutations is one of key technical issues. However, the risk is expected to be reduced by using more sophisticated enzymes [16,23,29,44], meticulous design of the guiding molecule [45], and prior genome-wide profiling of likely off-target effects of such nucleases [46,47]. Although mosaicism might be avoided by more careful consideration of the timing and method of microinjection, one report has demonstrated that the degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction [25]. Therefore, researchers may consider initiating human germline editing research if they have scientific or medical questions that can be addressed only by using the human germline. During the development of mitochondrial donation, human oocytes were fertilized by ICSI [48] or were parthenogenetically activated [49] to avoid the creation of human embryos for research. Alternatively, abnormally fertilized zygotes (which have recently been similarly used in the first human germline genome-editing research [41]) were employed [50]. Following nuclear genome transfer between oocytes or one-cell-stage embryos, the mitochondrial DNA profile was investigated in the blastocysts [48–50], established ESCs [48,49], and their differentiated cells [49]. Likewise, human germline genome-editing research may include the creation of human embryos, microinjection of programmable nucleases, embryo culture, subsequent
Efficiency in embryos a
NHEJ Monkey zygote
NR0B1, PPARG, RAG1
18.2–40.9% (single) 9.1–27.3% (double)
Monkey zygote
MECP2
Monkey zygote
MECP2
Bovine zygote Bovine zygote Ovine zygote Porcine zygote Porcine zygote
LDLR MSTN MSTN RELA NPC1
Porcine zygote Rat zygote
VWF IgM (Igh6)
Rat zygote
Tet1, Tet2, Tet3
Mouse zygote
Pibf1, Sepw1
Mouse zygote
Tet1, Tet2, Tet3, Sry, Uty
Mouse Mouse Mouse Mouse Mouse
Mecp2 Exo1 Fgf10 Fgf10 Tyr
zygote zygote zygote zygote zygote
Mouse zygote
Tyr, miR-205, Arf
Rat SSCs
Epsti1 and Erbb3
HDR Human tri-pronuclear zygote
Introduction of silent mutations into HBB
Rat zygote
Correction of Tyrc, Asipa, Kith
Efficiency in neonates b
Off-target mutation c
Platform
Delivery
Remarks
Ref.
No
Cas9
mRNA/sgRNA
[2]
9.5% (rhesus) 3.7% (cynomolgus)
No
TALENs
Plasmid
2.0%
N.D.
TALENs
mRNA
N.D. N.D. N.D. N.D. No
TALENs TALENs TALENs TALENs Cas9
mRNA mRNA mRNA mRNA mRNA/sgRNA
14.5% 3.9–5.5% (mRNA)
No No (plasmid) Yes (mRNA)
Cas9 TALENs
mRNA/sgRNA Plasmid or mRNA
14.3–18.8% (double; Tet1, Tet2) 18.6% (triple) 4.1–10.8% (Pibf1) 4.8% (Sepw1) 8.0–17.6% (single) 14.7–15.3% (double; Tet1, Tet2)
Yes (triple)
Cas9
mRNA/sgRNA
A set of twin female monkeys with modified RAG1 and PPARG were born Three miscarried rhesus and cynomolgus male fetuses had MECP2 mutations A modified male monkey appeared to be mosaic Cytoplasmic injection Cytoplasmic injection Cytoplasmic injection Cytoplasmic injection Mosaicism Cytoplasmic injection Cytoplasmic injection Mosaicism Plasmid, pronuclear mRNA, cytoplasmic injection Mosaicism Cytoplasmic injection
No (Pibf1)
TALENs
mRNA
No (Tet1, Tet2) N.D. (Tet3)
Cas9
mRNA/sgRNA
0–10.3% 14.3–41.7% 1.3–1.5% 3–50%
No N.D. N.D. N.D. N.D.
Cas9 nickase TALENs Cas9 TALENs Cas9
mRNA/sgRNA mRNA mRNA/sgRNA mRNA/sgRNA mRNA/sgRNA
2.8% (triple)
N.D.
TALENs
mRNA
N.D.
Cas9
Plasmid/gRNA
Yes
Cas9
mRNA/gRNA/ssODN
No
Cas9
mRNA/gRNA/ssODN
3.8% 18.8% 3.8% 0.5% 10.5%
80–100% d 1.4–6.8%
4.7%
7.7%(Tyr) 18.2%(Asip) 4.0%(Kit)
Mosaicism Cytoplasmic injection Pronuclear injection
Cytoplasmic injection Pronuclear injection Cytoplasmic injection Cytoplasmic injection Mosaicism Pronuclear or cytoplasmic injection Mosaicism Cytoplasmic injection Targeted mutations via NHEJ were confirmed in the offspring Cas9 and GFP mRNAs used Mosaicism Cytoplasmic injection Mosaicism Pronuclear injection
[3]
[37] [33] [36] [36] [33] [34] [35] [30]
[31]
[14] [15]
[16] [17] [18] [18] [19]
[20] [39]
[41]
[32]
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Targeted gene
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Germline
Opinion
Table 1. Examples of genetic modification of mammalian germline by genome editing
Targeted gene
Mouse zygote
Introduction of mCherry into Nanog, GFP into Oct4
Mouse zygote
Introduction of V5 into Sox2, lox into Mecp2 Correction of Crygc / Introduction of a STOP codon into Fah
Mouse zygote Mouse zygote
Mouse zygote Mouse zygote
Mouse zygote
Mouse zygote Mouse zygote Mouse zygote Mouse zygote SSCs
Efficiency in embryos a
6.0% (Sox2) 0.8% (Mecp2) 4.4–5.7% 2.0% (wild type) 2.0% (nickase mutant) 3.5% e
Correction of Cryb1rd8 Correction of Dmdmdx
Introduction of a single nucleotide mutation into Tyr Introduction of EGFP into 32 selected genes Introduction of TurboRFP or TagBFP into Rosa26 Introduction of FLAG-tag into Hprt Correction of Crygc /
16.7%
Delivery
Remarks
Ref.
Cas9
mRNA/sgRNA/plasmid
Cytoplasmic or pronuclear injection
[21]
Cas9
mRNA/sgRNA/ssODN
[21]
Cas9 Cas9 wild type and mutant
mRNA/sgRNA/ssODN mRNA/sgRNA/ssODN
Cytoplasmic or pronuclear injection Cytoplasmic injection Pronuclear injection
TALENs
mRNA/ssODN
No
Cas9
Plasmid/ssODN
injection
[26]
7.1%
Yes
Cas9
Plasmid
Pronuclear injection
[27]
0.5%
No
TALENs
mRNA/targeting vector
[28]
20%
No
Cas9 nickase
mRNA/sgRNA/ssODN
Mosaicism Cytoplasmic injection Cytoplasmic injection
No
Cas9
Plasmid/sgRNA/ssODN
Gene corrections via HDR or NHEJ and were confirmed in the offspring.
[38]
N.D.
Mito-TALENs
mRNA
Significant reduction of mtDNA copy number was confirmed Fertilization using the oocytes was not performed Cytoplasmic injection
[40]
Cas9
mRNA/sgRNA/ssODN
Genetically modified blastocysts per blastocyst which underwent Cas9 treatment (%).
Genetically modified neonates (including fetus) per injected zygote (%).
[24] injection [25] injection only, or and cytoplasmic
[29]
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d
[22] [23]
0.49%
No
Genetically modified neonates (including fetus) per transferred embryo (%), except underlined numbers (genetically modified neonates per neonates born).
N.D., not determined.
e
Yes
Platform
Genetically modified embryos per injected zygote (%).
b c
Off-target mutation c Yes (Nanog, Oct4) Yes (Mecp2) Yes Yes (mutant A and m.9176 Mouse oocytes T>C mutations fused with cells from patient
a
Efficiency in neonates b 1.7% (Nanog) 3.0% (Oct4)
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Table 1 (Continued )
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derivation of human embryonic stem cells (hESCs), and genetic and functional analyses of their differentiated cells (Figure 1). If human SSCs (hSSCs) are edited, the developmental potential of the resulting spermatozoa would need to be assessed, thus requiring the creation of human embryos for research. Regulation of human embryo research Researchers who conduct human germline genome-editing research must observe the regulations in force that govern human embryo research, and must obtain informed consent from the gamete or embryo donor. According to a recent regulatory survey of 17 countries (the full list is available in [51]) that allow hESC establishment from surplus embryos derived from ART, 15 of these countries permit the creation of embryos for research purposes under some circumstances. The statutes or guidelines of these countries restrict the culture period of the embryos to either the 14th day of embryo development or the formation of the primitive streak, which signifies the start of a unique human being. However, this culture period is sufficient to allow the establishment of hESCs. Although therapeutic cloning by somatic cell nuclear transfer is frequently listed as the purpose in these 15 countries, Belgium, Canada, Denmark, Japan, and the UK permit research that creates human embryos for improving or
Microinjecon of customized nucleases
providing instruction in ART. The indicated purpose potentially implies that germline genome-editing research may be permitted after prior consultation or permission from the authorities if the gene modification is associated with improving embryo viability, implantation, or the pregnancy rate. Most notably, the UK explicitly sanctions genetically modifying human embryos under the Human Fertilisation and Embryology Act (http://www.legislation. gov.uk/ukpga/2008/22/notes/division/6/1/11/8) if a license is obtained from the HFEA. Even if researchers do not have permission to create human embryos for research purposes, they can use existing embryos derived from surplus in vitro fertilization (IVF) embryos, or embryos screened out by preimplantation genetic diagnosis (PGD) [52] because of a genetic defect in the course of ART, although permission from the authorities and/or the approval of an institutional review board (IRB) is still required. Moreover, the Chinese guidelines permit parthenogenesis and the creation of human embryos for deriving hESCs if approved by an IRB [51]. It should be noted that the Cas9 research using abnormally fertilized zygotes was approved by an IRB and informed consent was obtained from the donors [41]. Furthermore, US researchers with nonfederal funding may be allowed to create human embryos for research in some states if an IRB and a stem cell oversight
Blastomere biopsy for PGD In nuclear genome
Culture
Culture Zygotes (one-cell-stage embryos)
Cleavage process (3 days postferlizaon)
E.g., homology directed repair
Blastocysts (4–5 days postferlizaon)
Out growth of inner cell mass Trophectoderm biopsy for PGD
Directed differenaon
Differenated cells for funconal analysis
Passages
Propagaon
Isolated cell line
Embryonic stem cells
TCAGTTTT CCC G
Genec analysis TRENDS in Molecular Medicine
Figure 1. A protocol for human germline genome-editing research. This research will likely begin by creating human embryos after the approval of an institutional review board (IRB) and/or health authority. Otherwise, researchers can use existing surplus embryos derived from assisted reproductive technology (ART) treatment cycles that were not to be used for assisted reproduction. Genome-editing nucleases are microinjected into the one-cell-stage embryos. The embryos are then cultured, and biopsied embryonic cells are subjected to genetic analyses, including preimplantation genetic diagnosis (PGD) [52], to investigate the gene modification. Subsequently, embryonic stem cells (ESCs) can be established if necessary. Genetic and functional analyses can be conducted using ESC-derived differentiated cells. If spermatogonial stem cells (SSCs) are edited, the developmental potential of the spermatozoa derived from the edited SSCs would then be assessed, thus requiring the creation of human embryos for research.
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committee approve the protocol [51]. Therefore, Chinese and some US researchers reside in a regulatory landscape that is permissive for human germline genome-editing research. Regulation of the clinical application of human germline genome editing Even if genome editing results in the introduction of a small indel in the human germline, the reproductive use of edited embryos or gametes is prohibited in many countries. A recent investigation revealed that 29 of 39 countries, by statute or guideline, ban human germline gene modification for reproductive purposes (Figure 2) [4]. However, the guidelines by which China, India, Ireland, and Japan ban human germline gene modification are less strictly enforced and are subject to amendment. Of the remaining ten countries, nine are ambiguous about this subject. The USA does not ban, but does restrict such clinical application by regulatory review of the FDA in addition to the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (2013) [4]. This regulatory landscape has been established based on the methodology of conventional genetic engineering – which frequently requires the use
of a drug-resistance or marker gene to facilitate screening for a rare desired mutant among a large excess of variants. In the genome-editing era, one may need to carefully interpret the provisions of the legislation governing human germline gene modification for reproductive purposes. For example, Belgium, Bulgaria, Canada, Denmark, Sweden, and the Czech Republic forbid germline gene modification on the grounds that a modified gene may be inherited by offspring or that the gene modification may impair human embryo development [4]. However, if genome editing can, without off-target mutations, completely correct a mutation in the germline genome, is this act still illegal in these countries? The resulting embryos or germ cells have a wild type genome which is likely to contribute to the viability of the embryo or fetus, or to the health of progeny. Human germline genome editing may be beyond the safety concerns addressed in the regulations in some countries. With the advent of genome editing, some countries with guidelines that ban or are ambiguous about germline gene modification may need to reconsider their legal status if they recognize the potential risk of abuse of this technology for non-medical purposes such as eugenics or enhancement. Other countries which explicitly ban germline gene
Key: Ban (legislaon) Ban (guidelines) Restricve Ambiguous
TRENDS in Molecular Medicine
Figure 2. The international regulatory landscape of human germline gene modification (permitted to reuse by the authors of [4]). Thirty-nine countries were investigated and categorized with regard to their view on germline gene modification. The different categories include ‘ban by legislation’ (25, pink), ‘ban by guidelines’ (four, faint pink), ‘ambiguous’ (nine, grey), and ‘restrictive’ (the USA, light-grey). The non-colored countries were excluded from this survey. See also the full list of the 39 countries shown in Table S1 in [4]. Note that the UK has legalized a form of germline gene modification (mitochondrial donation) in 2015 (effective in October, 2015) although the country is colored pink in this map.
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Opinion modification on the basis of modified genes should reconsider the provisions in their regulations given the potential for germline genome editing to cause social unrest. Potential socioethical implications Taking the regulatory approval of mitochondrial donation into consideration, the most likely purpose of human germline-editing research is to prevent definitive inheritance of a severe genetic disease by offspring [4]. Importantly, the results of the mito-TALEN research [40] suggest that this approach potentially bypasses one of the major ethical issues associated with mitochondrial donation, that is, the need for oocyte donation which might impose ovarian hyperstimulation syndrome on a specific haplogroup of females [6]. An alternative to the mito-TALENs might be a type of PGD which uses whole-genome amplification and the sequencing of polar bodies to identify oocytes with a low mtDNA mutation load [53]. However, this approach is limited to female patients or carriers whose oocytes have different levels of mtDNA mutation load, thereby enabling oocyte selection. If mito-TALEN reaches the level of clinical application, mitochondrial donation which requires oocyte donors will no longer be necessary for removing mtDNA mutations in oocytes from the patient. The purpose of preventive medicine can be applicable to an autosomal recessive disease where both parents are homozygous (e.g., cystic fibrosis [54]) provided that the prospective parents do not wish to use donor gametes to have children. In addition, autosomal dominant diseases in which one or both parents is/are homozygous (e.g., Huntington’s disease [55]) will also likely be considered. Correcting a gene mutation responsible for male infertility in germ cells (e.g., CFTR [56]) is another conceivable purpose. These conditions could be considered as one of the options open to patients should they wish to have genetically related children. As mentioned above, Cas9-mediated gene editing in human tri-pronuclear zygotes to repair mutations in HBB has already been reported [41]. Likewise, if human germline genome-editing research is conducted for preventing definitive inheritance of a serious genetic disease, such preventive medicine may relieve parents of the distress that stems from their genetic condition. Moreover, patients would also favorably view the treatment because a child born of germline genome editing and the subsequent generations would not be expected to develop the genetic disease. PGD has already been used clinically in some countries to screen out human embryos with mutations responsible for genetic conditions such as cystic fibrosis, spinal muscular atrophy, and late-onset Huntington disease [52]. The clinical use of PGD appears to justify germline genomeediting research because only embryos with no suspected mutation, but which have undergone the physical intervention of embryonic cell biopsy for genetic testing, are used for embryo transfer. In addition, embryonic genome editing might be the sole effective option to prevent the inheritance of a genetic disease in cases with a high hereditary probability of a disease mutation but where PGD is considered to be clinically inapplicable [54,55], although the risks of off-target mutations and mosaicism should be carefully assessed. Therefore, germline gene
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editing as a means to perform preventive medicine may be favorably considered in countries which have established regulations that authorize specific uses of PGD, including Belgium, the Czech Republic, Denmark, Finland, France, Greece, the Netherlands, Norway, Portugal, the Russian Federation, Serbia, Slovenia, Sweden, Spain, and the UK (http://www.coe.int/t/dg3/healthbioethic/Source/ INF_2010_6_dpidpn_en.pdf). Notably, Germany has recently amended its Embryo Protection Law and declared that PGD is, despite its fundamental prohibition, not illegal when performed according to the established strict regulations in force (http://www.drze.de/in-focus/ preimplantation-genetic-diagnosis/legal-aspects). By contrast, others may regard genome editing as a grave interference with human life – which they consider to begin at the point of fertilization. Others might judge that such a genetic intervention involving human embryos cannot be justified because the embryo is not an existing, affected individual. They would assert that medical treatment should be instigated at the onset of the disease after birth. Informed consent by prospective parent(s) is frequently obtained in the practice of ART [6]. However, some would suggest that parental consent alone is untenable because no prior consent can be obtained from a child who undergoes genome editing at the embryonic stage. Moreover, some opponents of germline genome editing suggest that PGD has created a bad precedent because the genetic test has been used for non-medical sex selection in some European countries, as well as in the USA [52] (http:// www.coe.int/t/dg3/healthbioethic/Source/INF_2010_6_ dpidpn_en.pdf). They would assert that germline genome editing for preventive medicine might lead to non-medical uses because genome-editing technology can readily be used not only for repairing a mutation but also for adding or disrupting a gene to rescue a mutant phenotype, potentially blurring the boundaries between medical and nonmedical use. Other opposition may be expected from the evolutionary point of view because this type of medicine implies an unprecedented event where a species corrects a mutation in the germline [4]. Concluding remarks and future perspectives Taking a retrospective view of the history of ART, Edwards et al. reported the results of IVF using human oocytes matured in vitro, with no attempt at pregnancy, in 1969 [57]. This research was subject to skepticism regarding the safety of the procedure and faced criticism on ethical grounds [10]. Sir Edwards sincerely engaged in open discussion about the social values of human embryo research [58]. Subsequent efforts to improve and optimize this research resulted in the birth of Louise Brown in 1978, the first child conceived through IVF. Although ethical concerns are still raised about ART, many infertile people have benefited from the development of modern ART. If the call for a moratorium [7] is intended to pause human germline-editing research (in addition to its clinical applications), the call appears to be too cautious about the socioethical implications of such research because many countries have already permitted human embryo research under relevant regulations [51] and have prohibited 7
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Opinion human germline gene modification for reproductive purposes [4]. If the calls [8,9] are intended to advocate that researchers are privileged to conduct human germlineediting research in a specific regulatory context, the outcomes of the research may, if not appropriately justified, lead the public towards absolute opposition to all in vitro experiments associated with human germline genome editing. If researchers believe that their research has sufficient justification to conduct human germline genome editing, they (excepting clinical applications) do not necessarily need to be subject to the moratorium when the research is to be conducted in countries where appropriate regulations are already in place. In such countries, researchers should fully discuss the socioethical implications of their research with the public. In this context, the reason(s) to conduct this research would preferably be medical – and not to enhance basic knowledge of the human germline [9]. Some researchers would explain their research from the perspective of future clinical applications, such as preventing the inheritance of genetic diseases before birth, as discussed in a recent movement on mitochondrial donation for mitochondrial diseases in the UK [6,11]. Other researchers may explain their research by focusing on understanding the molecular mechanisms underlying the onset of congenital anomalies. As a result, some research will be supported by society, while other projects will be discouraged or strictly regulated by newly established legislation. Although creating forums by experts, and holding international conferences, are both important [8], public dialogue will be vital to shape the socially-acceptable uses of germline genome editing (if any) and to avoid potential abuse of this technology. Acknowledgments The author thanks Motoko Araki for her help in the preparation of the figures. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant 26460586 (T.I.).
References 1 Sasaki, E. et al. (2009) Generation of transgenic non-human primates with germline transmission. Nature 459, 523–527 2 Niu, Y. et al. (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156, 836–843 3 Liu, H. et al. (2014) TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell 14, 323–328 4 Araki, M. and Ishii, T. (2014) International regulatory landscape and integration of corrective genome editing into in vitro fertilization. Reprod. Biol. Endocrinol. 12, 108 5 Frankel, M. and Chapman, A. (2000) Human Inheritable Genetic Modifications. Assessing Scientific, Ethical, Religious and Policy Issues, American Association for the Advancement of Sciences 6 Ishii, T. (2014) Potential impact of human mitochondrial replacement on global policy regarding germline gene modification. Reprod. Biomed. Online 29, 150–155 7 Lanphier, E. et al. (2015) Don’t edit the human germ line. Nature 519, 410–411 8 Baltimore, D. et al. (2015) A prudent path forward for genomic engineering and germline gene modification. Science 348, 36–38 9 ISSCR (2015) The ISSCR Statement on Human Germline Genome Modification, International Society for Stem Cell Research 10 Johnson, M.H. et al. (2010) Why the Medical Research Council refused Robert Edwards and Patrick Steptoe support for research on human conception in 1971. Hum. Reprod. 25, 2157–2174 8
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11 UK Government (2015) The Human Fertilisation and Embryology (Mitochondrial Donation) Regulations 2015 (coming into force on October 29, 2015), The National Archives 12 Kim, H. and Kim, J.S. (2014) A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 13 Palermo, G.D. et al. (1995) Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil. Steril. 63, 1231– 1240 14 Sung, Y.H. et al. (2013) Knockout mice created by TALEN-mediated gene targeting. Nat. Biotechnol. 31, 23–24 15 Wang, H. et al. (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 16 Ran, F.A. et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 17 Mashimo, T. et al. (2013) Efficient gene targeting by TAL effector nucleases coinjected with exonucleases in zygotes. Sci. Rep. 3, 1253 18 Yasue, A. et al. (2014) Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems. Sci. Rep. 4, 5705 19 Yen, S.T. et al. (2014) Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev. Biol. 393, 3–9 20 Li, C. et al. (2014) Simultaneous gene editing by injection of mRNAs encoding transcription activator-like effector nucleases into mouse zygotes. Mol. Cell. Biol. 34, 1649–1658 21 Yang, H. et al. (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 22 Wu, Y. et al. (2013) Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell 13, 659–662 23 Li, F. et al. (2014) Efficient genetic manipulation of the NOD Rag1 / IL2RgammaC-null mouse by combining in vitro fertilization and CRISPR/Cas9 technology. Sci. Rep. 4, 5290 24 Low, B.E. et al. (2014) Correction of the Crb1rd8 allele and retinal phenotype in C57BL/6N mice via TALEN-mediated homology-directed repair. Invest. Ophthalmol. Vis. Sci. 55, 387–395 25 Long, C. et al. (2014) Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345, 1184– 1188 26 Mizuno, S. et al. (2014) Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system. Mamm. Genome 25, 327–334 27 Mashiko, D. et al. (2014) Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. Dev. Growth Differ. 56, 122–129 28 Kasparek, P. et al. (2014) Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases. FEBS Lett. 588, 3982–3988 29 Fujii, W. et al. (2014) Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system. Biochem. Biophys. Res. Commun. 445, 791–794 30 Tesson, L. et al. (2011) Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695–696 31 Li, W. et al. (2013) Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat. Biotechnol. 31, 684–686 32 Yoshimi, K. et al. (2014) Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform. Nat. Commun. 5, 4240 33 Carlson, D.F. et al. (2012) Efficient TALEN-mediated gene knockout in livestock. Proc. Natl. Acad. Sci. U.S.A. 109, 17382–17387 34 Wang, Y. et al. (2015) Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA. Sci. Rep. 5, 8256 35 Hai, T. et al. (2014) One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res. 24, 372–375 36 Proudfoot, C. et al. (2015) Genome edited sheep and cattle. Transgenic Res. 24, 147–153 37 Liu, Z. et al. (2014) Generation of a monkey with MECP2 mutations by TALEN-based gene targeting. Neurosci. Bull. 30, 381–386 38 Wu, Y. et al. (2015) Correction of a genetic disease by CRISPR-Cas9mediated gene editing in mouse spermatogonial stem cells. Cell Res. 25, 67–79
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Opinion 39 Chapman, K.M. et al. (2015) Targeted germline modifications in rats ising CRISPR/Cas9 and spermatogonial stem cells. Cell Rep. 10, 1828–1835 40 Reddy, P. et al. (2015) Selective elimination of mitochondrial mutations in the germline by genome editing. Cell 161, 459–469 41 Liang, P. et al. (2015) CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6, 363–372 42 Gaj, T. et al. (2012) Targeted gene knockout by direct delivery of zincfinger nuclease proteins. Nat. Methods 9, 805–807 43 Kim, S. et al. (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 44 Tsai, S.Q. et al. (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 45 Fu, Y. et al. (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 46 Kim, D. et al. (2015) Digenome-seq: genome-wide profiling of CRISPRCas9 off-target effects in human cells. Nat. Methods 12, 237–243 47 Tsai, S.Q. and Zheng, Z. (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 48 Tachibana, M. et al. (2013) Towards germline gene therapy of inherited mitochondrial diseases. Nature 493, 627–631 49 Paull, D. et al. (2013) Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants. Nature 493, 632–637 50 Craven, L. et al. (2010) Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465, 82–85 51 Ishii, T. et al. (2013) Ethical and legal issues arising in research on inducing human germ cells from pluripotent stem cells. Cell Stem Cell 13, 145–148
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52 Stern, H.J. (2014) Preimplantation genetic diagnosis: prenatal testing for embryos finally achieving its potential. J. Clin. Med. 3, 280–309 53 Hou, Y. et al. (2013) Genome analyses of single human oocytes. Cell 155, 1492–1506 54 Neocleous, V. et al. (2014) Apparent homozygosity of p.Phe508del in CFTR due to a large gene deletion of exons 4–11. Case Rep. Genet. 2014, 613863 55 Squitieri, F. et al. (2003) Homozygosity for CAG mutation in Huntington disease is associated with a more severe clinical course. Brain 126, 946–955 56 Chen, H. et al. (2012) Regulation of male fertility by CFTR and implications in male infertility. Hum. Reprod. Update 18, 703–713 57 Edwards, R.G. et al. (1969) Early stages of fertilization in vitro of human oocytes matured in vitro. Nature 221, 632–635 58 Edwards, R.G. and Sharpe, D.J. (1971) Social values and research in human embryology. Nature 231, 87–91 59 Flood, J.T. et al. (1990) Ooplasmic transfusion: prophase germinal vesicle oocytes made developmentally competent by microinjection of metaphase II egg cytoplasm. Fertil. Steril. 53, 1049–1054 60 Levron, J. et al. (1996) The development of mouse zygotes after fusion with synchronous and asynchronous cytoplasm. Hum. Reprod. 11, 1287–1292 61 Cohen, J. et al. (1997) Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet 350, 186–187 62 Barritt, J.A. et al. (2001) Mitochondria in human offspring derived from ooplasmic transplantation. Hum. Reprod. 16, 513–516 63 Barritt, J.A. et al. (2001) Rebuttal: interooplasmic transfers in humans. Reprod. Biomed. Online 3, 47–48 64 Zhang, J. et al. (2003) Pregnancy derived from human nuclear transfer. Fertil. Steril. 80, 56
9
Opinion
Germline genome-editing research and its socioethical implications Tetsuya Ishii Office of Health and Safety, Hokkaido University, Sapporo 060-0808, Hokkaido, Japan
Genetically modifying eggs, sperm, and zygotes (‘germline’ modification) can impact on the entire body of the resulting individual and on subsequent generations. With the advent of genome-editing technology, human germline gene modification is no longer theoretical. Owing to increasing concerns about human germline gene modification, a voluntary moratorium on human genome-editing research and/or the clinical application of human germline genome editing has recently been called for. However, whether such research should be suspended or encouraged warrants careful consideration. The present article reviews recent research on mammalian germline genome editing, discusses the importance of public dialogue on the socioethical implications of human germline genome-editing research, and considers the relevant guidelines and legislation in different countries. Urgent calls for a moratorium on human germline genome editing Rapid technological advances in the past decade have enabled mammalian germline gene modification. Notably, injecting a self-inactivating lentiviral vector into non-human primate embryos resulted in the generation of transgenic monkeys from which germline transmission was attained [1]. However, this methodology requires viral vectors which are frequently difficult to handle in the laboratory, and is largely limited to gene addition. More recently, genome editing, including the use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/ Cas (CRISPR-associated) systems such as Cas9, has demonstrated efficient genetic modification in non-human primates [2,3] as well as in human embryonic stem cells (hESCs) (reviewed in [4]). Although mammalian cells have been frequently been transduced by either integrating or non-integrating viral vectors harboring genome-editing nucleases, customized nucleases can, in some cases, be introduced into cells as an RNA or protein, together with a DNA template (if needed). More importantly, these programmable nucleases, particularly Cas9, are much easier to use for various types of gene Corresponding author: Ishii, T. ([email protected]). Keywords: germline genome editing; CRISPR/Cas9; ethics; genome engineering; embryonic stem cells. 1471-4914/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2015.05.006
modification, and thus are now being used instead of conventional genetic engineering in many laboratories worldwide. The robustness of this genome engineering technology has made it conceivable that gene modification of the human germline (oocytes, sperm, zygotes, and embryos) (Box 1) is becoming feasible in the clinical setting [4]. However, this type of gene modification has raised tremendous debate in the context of medical beneficence, safety concerns, challenges to human dignity, and risk of abuse for eugenics or enhancement (the parental pursuit of specific traits for non-medical reasons) [5]. Consequently, many countries forbid human germline gene modification for reproductive purposes [4,6]. Recently, representatives of the Alliance for Regenerative Medicine, a group of interested stakeholders including Cas9 developers and the International Society for Stem Cell Research (ISSCR), have called for a voluntary moratorium on research into and/or the clinical application of human germline genome editing owing to increasing bioethical concern [7–9]. These calls could be welcome because a moratorium might provide opportunities to reach a domestic or international consensus regarding whether society accepts or rejects human germline gene modification for medical purposes. The first call asked researchers not to carry out either research in the field or clinical application of the findings [7]. The latter two calls requested a moratorium on the clinical application of genome engineering, but suggested that in vitro research on editing the human germline genome should be permitted [8,9]. However, as demonstrated by the history of assisted reproductive technology (ART), the outcomes of human embryo research send a strong message to the public [10]. Meanwhile, draft regulations allowing mitochondrial donation, which changes the mitochondrial DNA content by nuclear transfer between oocytes or embryos to prevent maternal transmission of serious mitochondrial diseases, have recently been approved in the UK (effective on October 29, 2015) [11]. Although a controversial germline gene modification that has garnered intense attention worldwide – from initial consideration by the Human Fertilisation and Embryology Authority (HFEA) to the vote in Parliament – the UK became the first nation to legalize a form of human germline gene modification, changing the international regulatory landscape [6]. This opinion article reviews recent developments in mammalian germline genome-editing research. In addition, the significance of a public dialogue regarding the socioethical implications of Trends in Molecular Medicine xx (2015) 1–9
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Opinion Box 1. Human germline gene modification and clinical cases In a sexually reproducing organism, the genetic information for an individual is derived from oocytes and spermatozoa. Genetically modifying germ cells or zygotes (one-cell-stage embryos) can impact on the entire body of the offspring, and on subsequent generations via modified germ cells. Therefore, germline gene modification has been considered to be efficacious against genetic diseases. Two forms of germline gene modification focused on mitochondria (that contain their own genome) have been performed for treating infertility in the clinical setting. Case 1. Ooplasmic transfer Ooplasmic transfer is technically based on intracytoplasmic sperm injection (ICSI) for treating male infertility [13]. A small portion of the cytoplasm from donor oocytes (ooplasm) is transferred, together with a spermatozoon, into quality-compromised oocytes to restore their developmental potential. The mitochondria in the ooplasm were considered to be one of the key factors involved in the improved fertility in animal studies [59,60]. Cohen et al. reported the first case of pregnancy and childbirth in 1997 [61]. With the consent of the donors, this technique was applied in the USA. Heteroplasmy was confirmed in the blood of two infants born via this technique [62]. However, two congenital anomalies, 45 XO (Turner syndrome) and pervasive developmental disorder (PDD), were found in other children [63]. Subsequently, the FDA decided to regulate this treatment owing to the potential health risk to offspring. Meanwhile, ooplasmic transfer had already assisted nearly 30 childbirths worldwide [62]. In 2014, Alana Saarinen was introduced as one of the offspring (http://www.bbc.com/news/ magazine-28986843). She appeared to be healthy. Case 2. Pronuclear transfer Pronuclear transfer aims to exchange the pronuclei between two different one-cell-stage embryos. In 2003, Zhang et al. reported the first case of a pregnancy following pronuclear transfer carried out in China [64]. The transfer of the five treated embryos resulted in a triplet pregnancy with fetal heartbeats. A fetus was reduced for better development of the other two. However, these fetuses died of respiratory distress and cord prolapse, respectively. The mitochondrial DNA profiles of the fetal cells were similar to those from the donor. It was concluded that pregnancy with a normal karyotype is attainable by pronuclear transfer. However, the use of this procedure resulted in the establishment of the Guidelines on Human Assisted Reproductive Technologies (2003), which forbid using ooplasmic or nuclear transfer as well as genetic modification of the germline for reproductive purposes [4].
in vitro research on editing the human germline genome is discussed, while reviewing the guidelines and legislation that govern human embryo research, as well as reproduction following germline gene modification, in different countries across the world. Genome editing-mediated germline gene modification Genome editing can directly modify a gene within the genome, while conventional genetic engineering involves introducing an additional gene copy (reviewed in [12]). Introducing bacterial nucleases customized to home to a specific sequence into cells or embryos initiates a desired genetic change by creating double-strand breaks (DSBs) in the target sequence. Subsequently, the DSBs are repaired via non-homologous end-joining (NHEJ) in the absence of a repair template, or by homology-directed repair (HDR) if provided with a template. The NHEJ pathway efficiently results in a small insertion or deletion (termed ‘indel’) at a target site, whereas HDR can repair a mutation or add a desired DNA sequence at a specific site. 2
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For germline gene modification, the programmable nucleases (together with single guide RNAs in the case of Cas9) are directly injected into animal one-cell-stage embryos (reviewed in [4]). Embryo microinjection – which is outwardly similar to a common ART technique, intracytoplasmic sperm injection (ICSI) [13] – demonstrated efficient generation of genetically modified animals including mice [14–29], rats [30–32], pigs [33–35], sheep [36], cattle [33,36], and non-human primates [2,3,37], via NHEJ or the HDR pathway (Table 1). Moreover, germline gene modifications are also attainable via genome editing of spermatogonial stem cells (SSCs), potentially preventing mosaicism in offspring by avoiding the totipotent states that accompany embryogenesis. Successful editing using the SSC approach has been demonstrated in mice [38] and in rats [39]. Of note, Reddy et al. demonstrated that mitochondria-targeted TALENs (mito-TALENs) can be used in mouse oocytes fused with cells from a patient to eliminate human mitochondrial DNA (mtDNA) mutations [40]. More recently, Cas9-mediated gene modification was reported using human tri-pronuclear zygotes [41] (Table 1). However, the efficiency of HDR of the b-globin gene (HBB) was 4.7% (per injected zygote) and the modified embryos displayed mosaicism in which wild type cells and genetically modified cells coexisted. This latter might be due to the use of abnormally fertilized embryos. In addition, Cas9 treatment apparently induced off-target mutations. These results suggest that there is room to optimize the protocol for germline genome editing in terms of (i) the methodology used to introduce the nucleases (the dose and the form of the enzymes – plasmid, mRNA, or protein [42,43]; cytoplasmic or pronuclear injection), (ii) mosaicism [14,19,20,24,25,28,30–32,34,37], and (iii) possible off-target mutations [21–24,27,30,31] (Table 1). The risk of offtarget mutations is one of key technical issues. However, the risk is expected to be reduced by using more sophisticated enzymes [16,23,29,44], meticulous design of the guiding molecule [45], and prior genome-wide profiling of likely off-target effects of such nucleases [46,47]. Although mosaicism might be avoided by more careful consideration of the timing and method of microinjection, one report has demonstrated that the degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction [25]. Therefore, researchers may consider initiating human germline editing research if they have scientific or medical questions that can be addressed only by using the human germline. During the development of mitochondrial donation, human oocytes were fertilized by ICSI [48] or were parthenogenetically activated [49] to avoid the creation of human embryos for research. Alternatively, abnormally fertilized zygotes (which have recently been similarly used in the first human germline genome-editing research [41]) were employed [50]. Following nuclear genome transfer between oocytes or one-cell-stage embryos, the mitochondrial DNA profile was investigated in the blastocysts [48–50], established ESCs [48,49], and their differentiated cells [49]. Likewise, human germline genome-editing research may include the creation of human embryos, microinjection of programmable nucleases, embryo culture, subsequent
Efficiency in embryos a
NHEJ Monkey zygote
NR0B1, PPARG, RAG1
18.2–40.9% (single) 9.1–27.3% (double)
Monkey zygote
MECP2
Monkey zygote
MECP2
Bovine zygote Bovine zygote Ovine zygote Porcine zygote Porcine zygote
LDLR MSTN MSTN RELA NPC1
Porcine zygote Rat zygote
VWF IgM (Igh6)
Rat zygote
Tet1, Tet2, Tet3
Mouse zygote
Pibf1, Sepw1
Mouse zygote
Tet1, Tet2, Tet3, Sry, Uty
Mouse Mouse Mouse Mouse Mouse
Mecp2 Exo1 Fgf10 Fgf10 Tyr
zygote zygote zygote zygote zygote
Mouse zygote
Tyr, miR-205, Arf
Rat SSCs
Epsti1 and Erbb3
HDR Human tri-pronuclear zygote
Introduction of silent mutations into HBB
Rat zygote
Correction of Tyrc, Asipa, Kith
Efficiency in neonates b
Off-target mutation c
Platform
Delivery
Remarks
Ref.
No
Cas9
mRNA/sgRNA
[2]
9.5% (rhesus) 3.7% (cynomolgus)
No
TALENs
Plasmid
2.0%
N.D.
TALENs
mRNA
N.D. N.D. N.D. N.D. No
TALENs TALENs TALENs TALENs Cas9
mRNA mRNA mRNA mRNA mRNA/sgRNA
14.5% 3.9–5.5% (mRNA)
No No (plasmid) Yes (mRNA)
Cas9 TALENs
mRNA/sgRNA Plasmid or mRNA
14.3–18.8% (double; Tet1, Tet2) 18.6% (triple) 4.1–10.8% (Pibf1) 4.8% (Sepw1) 8.0–17.6% (single) 14.7–15.3% (double; Tet1, Tet2)
Yes (triple)
Cas9
mRNA/sgRNA
A set of twin female monkeys with modified RAG1 and PPARG were born Three miscarried rhesus and cynomolgus male fetuses had MECP2 mutations A modified male monkey appeared to be mosaic Cytoplasmic injection Cytoplasmic injection Cytoplasmic injection Cytoplasmic injection Mosaicism Cytoplasmic injection Cytoplasmic injection Mosaicism Plasmid, pronuclear mRNA, cytoplasmic injection Mosaicism Cytoplasmic injection
No (Pibf1)
TALENs
mRNA
No (Tet1, Tet2) N.D. (Tet3)
Cas9
mRNA/sgRNA
0–10.3% 14.3–41.7% 1.3–1.5% 3–50%
No N.D. N.D. N.D. N.D.
Cas9 nickase TALENs Cas9 TALENs Cas9
mRNA/sgRNA mRNA mRNA/sgRNA mRNA/sgRNA mRNA/sgRNA
2.8% (triple)
N.D.
TALENs
mRNA
N.D.
Cas9
Plasmid/gRNA
Yes
Cas9
mRNA/gRNA/ssODN
No
Cas9
mRNA/gRNA/ssODN
3.8% 18.8% 3.8% 0.5% 10.5%
80–100% d 1.4–6.8%
4.7%
7.7%(Tyr) 18.2%(Asip) 4.0%(Kit)
Mosaicism Cytoplasmic injection Pronuclear injection
Cytoplasmic injection Pronuclear injection Cytoplasmic injection Cytoplasmic injection Mosaicism Pronuclear or cytoplasmic injection Mosaicism Cytoplasmic injection Targeted mutations via NHEJ were confirmed in the offspring Cas9 and GFP mRNAs used Mosaicism Cytoplasmic injection Mosaicism Pronuclear injection
[3]
[37] [33] [36] [36] [33] [34] [35] [30]
[31]
[14] [15]
[16] [17] [18] [18] [19]
[20] [39]
[41]
[32]
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Targeted gene
3
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Germline
Opinion
Table 1. Examples of genetic modification of mammalian germline by genome editing
Targeted gene
Mouse zygote
Introduction of mCherry into Nanog, GFP into Oct4
Mouse zygote
Introduction of V5 into Sox2, lox into Mecp2 Correction of Crygc / Introduction of a STOP codon into Fah
Mouse zygote Mouse zygote
Mouse zygote Mouse zygote
Mouse zygote
Mouse zygote Mouse zygote Mouse zygote Mouse zygote SSCs
Efficiency in embryos a
6.0% (Sox2) 0.8% (Mecp2) 4.4–5.7% 2.0% (wild type) 2.0% (nickase mutant) 3.5% e
Correction of Cryb1rd8 Correction of Dmdmdx
Introduction of a single nucleotide mutation into Tyr Introduction of EGFP into 32 selected genes Introduction of TurboRFP or TagBFP into Rosa26 Introduction of FLAG-tag into Hprt Correction of Crygc /
16.7%
Delivery
Remarks
Ref.
Cas9
mRNA/sgRNA/plasmid
Cytoplasmic or pronuclear injection
[21]
Cas9
mRNA/sgRNA/ssODN
[21]
Cas9 Cas9 wild type and mutant
mRNA/sgRNA/ssODN mRNA/sgRNA/ssODN
Cytoplasmic or pronuclear injection Cytoplasmic injection Pronuclear injection
TALENs
mRNA/ssODN
No
Cas9
Plasmid/ssODN
injection
[26]
7.1%
Yes
Cas9
Plasmid
Pronuclear injection
[27]
0.5%
No
TALENs
mRNA/targeting vector
[28]
20%
No
Cas9 nickase
mRNA/sgRNA/ssODN
Mosaicism Cytoplasmic injection Cytoplasmic injection
No
Cas9
Plasmid/sgRNA/ssODN
Gene corrections via HDR or NHEJ and were confirmed in the offspring.
[38]
N.D.
Mito-TALENs
mRNA
Significant reduction of mtDNA copy number was confirmed Fertilization using the oocytes was not performed Cytoplasmic injection
[40]
Cas9
mRNA/sgRNA/ssODN
Genetically modified blastocysts per blastocyst which underwent Cas9 treatment (%).
Genetically modified neonates (including fetus) per injected zygote (%).
[24] injection [25] injection only, or and cytoplasmic
[29]
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d
[22] [23]
0.49%
No
Genetically modified neonates (including fetus) per transferred embryo (%), except underlined numbers (genetically modified neonates per neonates born).
N.D., not determined.
e
Yes
Platform
Genetically modified embryos per injected zygote (%).
b c
Off-target mutation c Yes (Nanog, Oct4) Yes (Mecp2) Yes Yes (mutant A and m.9176 Mouse oocytes T>C mutations fused with cells from patient
a
Efficiency in neonates b 1.7% (Nanog) 3.0% (Oct4)
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Table 1 (Continued )
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derivation of human embryonic stem cells (hESCs), and genetic and functional analyses of their differentiated cells (Figure 1). If human SSCs (hSSCs) are edited, the developmental potential of the resulting spermatozoa would need to be assessed, thus requiring the creation of human embryos for research. Regulation of human embryo research Researchers who conduct human germline genome-editing research must observe the regulations in force that govern human embryo research, and must obtain informed consent from the gamete or embryo donor. According to a recent regulatory survey of 17 countries (the full list is available in [51]) that allow hESC establishment from surplus embryos derived from ART, 15 of these countries permit the creation of embryos for research purposes under some circumstances. The statutes or guidelines of these countries restrict the culture period of the embryos to either the 14th day of embryo development or the formation of the primitive streak, which signifies the start of a unique human being. However, this culture period is sufficient to allow the establishment of hESCs. Although therapeutic cloning by somatic cell nuclear transfer is frequently listed as the purpose in these 15 countries, Belgium, Canada, Denmark, Japan, and the UK permit research that creates human embryos for improving or
Microinjecon of customized nucleases
providing instruction in ART. The indicated purpose potentially implies that germline genome-editing research may be permitted after prior consultation or permission from the authorities if the gene modification is associated with improving embryo viability, implantation, or the pregnancy rate. Most notably, the UK explicitly sanctions genetically modifying human embryos under the Human Fertilisation and Embryology Act (http://www.legislation. gov.uk/ukpga/2008/22/notes/division/6/1/11/8) if a license is obtained from the HFEA. Even if researchers do not have permission to create human embryos for research purposes, they can use existing embryos derived from surplus in vitro fertilization (IVF) embryos, or embryos screened out by preimplantation genetic diagnosis (PGD) [52] because of a genetic defect in the course of ART, although permission from the authorities and/or the approval of an institutional review board (IRB) is still required. Moreover, the Chinese guidelines permit parthenogenesis and the creation of human embryos for deriving hESCs if approved by an IRB [51]. It should be noted that the Cas9 research using abnormally fertilized zygotes was approved by an IRB and informed consent was obtained from the donors [41]. Furthermore, US researchers with nonfederal funding may be allowed to create human embryos for research in some states if an IRB and a stem cell oversight
Blastomere biopsy for PGD In nuclear genome
Culture
Culture Zygotes (one-cell-stage embryos)
Cleavage process (3 days postferlizaon)
E.g., homology directed repair
Blastocysts (4–5 days postferlizaon)
Out growth of inner cell mass Trophectoderm biopsy for PGD
Directed differenaon
Differenated cells for funconal analysis
Passages
Propagaon
Isolated cell line
Embryonic stem cells
TCAGTTTT CCC G
Genec analysis TRENDS in Molecular Medicine
Figure 1. A protocol for human germline genome-editing research. This research will likely begin by creating human embryos after the approval of an institutional review board (IRB) and/or health authority. Otherwise, researchers can use existing surplus embryos derived from assisted reproductive technology (ART) treatment cycles that were not to be used for assisted reproduction. Genome-editing nucleases are microinjected into the one-cell-stage embryos. The embryos are then cultured, and biopsied embryonic cells are subjected to genetic analyses, including preimplantation genetic diagnosis (PGD) [52], to investigate the gene modification. Subsequently, embryonic stem cells (ESCs) can be established if necessary. Genetic and functional analyses can be conducted using ESC-derived differentiated cells. If spermatogonial stem cells (SSCs) are edited, the developmental potential of the spermatozoa derived from the edited SSCs would then be assessed, thus requiring the creation of human embryos for research.
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committee approve the protocol [51]. Therefore, Chinese and some US researchers reside in a regulatory landscape that is permissive for human germline genome-editing research. Regulation of the clinical application of human germline genome editing Even if genome editing results in the introduction of a small indel in the human germline, the reproductive use of edited embryos or gametes is prohibited in many countries. A recent investigation revealed that 29 of 39 countries, by statute or guideline, ban human germline gene modification for reproductive purposes (Figure 2) [4]. However, the guidelines by which China, India, Ireland, and Japan ban human germline gene modification are less strictly enforced and are subject to amendment. Of the remaining ten countries, nine are ambiguous about this subject. The USA does not ban, but does restrict such clinical application by regulatory review of the FDA in addition to the National Institutes of Health (NIH) Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (2013) [4]. This regulatory landscape has been established based on the methodology of conventional genetic engineering – which frequently requires the use
of a drug-resistance or marker gene to facilitate screening for a rare desired mutant among a large excess of variants. In the genome-editing era, one may need to carefully interpret the provisions of the legislation governing human germline gene modification for reproductive purposes. For example, Belgium, Bulgaria, Canada, Denmark, Sweden, and the Czech Republic forbid germline gene modification on the grounds that a modified gene may be inherited by offspring or that the gene modification may impair human embryo development [4]. However, if genome editing can, without off-target mutations, completely correct a mutation in the germline genome, is this act still illegal in these countries? The resulting embryos or germ cells have a wild type genome which is likely to contribute to the viability of the embryo or fetus, or to the health of progeny. Human germline genome editing may be beyond the safety concerns addressed in the regulations in some countries. With the advent of genome editing, some countries with guidelines that ban or are ambiguous about germline gene modification may need to reconsider their legal status if they recognize the potential risk of abuse of this technology for non-medical purposes such as eugenics or enhancement. Other countries which explicitly ban germline gene
Key: Ban (legislaon) Ban (guidelines) Restricve Ambiguous
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Figure 2. The international regulatory landscape of human germline gene modification (permitted to reuse by the authors of [4]). Thirty-nine countries were investigated and categorized with regard to their view on germline gene modification. The different categories include ‘ban by legislation’ (25, pink), ‘ban by guidelines’ (four, faint pink), ‘ambiguous’ (nine, grey), and ‘restrictive’ (the USA, light-grey). The non-colored countries were excluded from this survey. See also the full list of the 39 countries shown in Table S1 in [4]. Note that the UK has legalized a form of germline gene modification (mitochondrial donation) in 2015 (effective in October, 2015) although the country is colored pink in this map.
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Opinion modification on the basis of modified genes should reconsider the provisions in their regulations given the potential for germline genome editing to cause social unrest. Potential socioethical implications Taking the regulatory approval of mitochondrial donation into consideration, the most likely purpose of human germline-editing research is to prevent definitive inheritance of a severe genetic disease by offspring [4]. Importantly, the results of the mito-TALEN research [40] suggest that this approach potentially bypasses one of the major ethical issues associated with mitochondrial donation, that is, the need for oocyte donation which might impose ovarian hyperstimulation syndrome on a specific haplogroup of females [6]. An alternative to the mito-TALENs might be a type of PGD which uses whole-genome amplification and the sequencing of polar bodies to identify oocytes with a low mtDNA mutation load [53]. However, this approach is limited to female patients or carriers whose oocytes have different levels of mtDNA mutation load, thereby enabling oocyte selection. If mito-TALEN reaches the level of clinical application, mitochondrial donation which requires oocyte donors will no longer be necessary for removing mtDNA mutations in oocytes from the patient. The purpose of preventive medicine can be applicable to an autosomal recessive disease where both parents are homozygous (e.g., cystic fibrosis [54]) provided that the prospective parents do not wish to use donor gametes to have children. In addition, autosomal dominant diseases in which one or both parents is/are homozygous (e.g., Huntington’s disease [55]) will also likely be considered. Correcting a gene mutation responsible for male infertility in germ cells (e.g., CFTR [56]) is another conceivable purpose. These conditions could be considered as one of the options open to patients should they wish to have genetically related children. As mentioned above, Cas9-mediated gene editing in human tri-pronuclear zygotes to repair mutations in HBB has already been reported [41]. Likewise, if human germline genome-editing research is conducted for preventing definitive inheritance of a serious genetic disease, such preventive medicine may relieve parents of the distress that stems from their genetic condition. Moreover, patients would also favorably view the treatment because a child born of germline genome editing and the subsequent generations would not be expected to develop the genetic disease. PGD has already been used clinically in some countries to screen out human embryos with mutations responsible for genetic conditions such as cystic fibrosis, spinal muscular atrophy, and late-onset Huntington disease [52]. The clinical use of PGD appears to justify germline genomeediting research because only embryos with no suspected mutation, but which have undergone the physical intervention of embryonic cell biopsy for genetic testing, are used for embryo transfer. In addition, embryonic genome editing might be the sole effective option to prevent the inheritance of a genetic disease in cases with a high hereditary probability of a disease mutation but where PGD is considered to be clinically inapplicable [54,55], although the risks of off-target mutations and mosaicism should be carefully assessed. Therefore, germline gene
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editing as a means to perform preventive medicine may be favorably considered in countries which have established regulations that authorize specific uses of PGD, including Belgium, the Czech Republic, Denmark, Finland, France, Greece, the Netherlands, Norway, Portugal, the Russian Federation, Serbia, Slovenia, Sweden, Spain, and the UK (http://www.coe.int/t/dg3/healthbioethic/Source/ INF_2010_6_dpidpn_en.pdf). Notably, Germany has recently amended its Embryo Protection Law and declared that PGD is, despite its fundamental prohibition, not illegal when performed according to the established strict regulations in force (http://www.drze.de/in-focus/ preimplantation-genetic-diagnosis/legal-aspects). By contrast, others may regard genome editing as a grave interference with human life – which they consider to begin at the point of fertilization. Others might judge that such a genetic intervention involving human embryos cannot be justified because the embryo is not an existing, affected individual. They would assert that medical treatment should be instigated at the onset of the disease after birth. Informed consent by prospective parent(s) is frequently obtained in the practice of ART [6]. However, some would suggest that parental consent alone is untenable because no prior consent can be obtained from a child who undergoes genome editing at the embryonic stage. Moreover, some opponents of germline genome editing suggest that PGD has created a bad precedent because the genetic test has been used for non-medical sex selection in some European countries, as well as in the USA [52] (http:// www.coe.int/t/dg3/healthbioethic/Source/INF_2010_6_ dpidpn_en.pdf). They would assert that germline genome editing for preventive medicine might lead to non-medical uses because genome-editing technology can readily be used not only for repairing a mutation but also for adding or disrupting a gene to rescue a mutant phenotype, potentially blurring the boundaries between medical and nonmedical use. Other opposition may be expected from the evolutionary point of view because this type of medicine implies an unprecedented event where a species corrects a mutation in the germline [4]. Concluding remarks and future perspectives Taking a retrospective view of the history of ART, Edwards et al. reported the results of IVF using human oocytes matured in vitro, with no attempt at pregnancy, in 1969 [57]. This research was subject to skepticism regarding the safety of the procedure and faced criticism on ethical grounds [10]. Sir Edwards sincerely engaged in open discussion about the social values of human embryo research [58]. Subsequent efforts to improve and optimize this research resulted in the birth of Louise Brown in 1978, the first child conceived through IVF. Although ethical concerns are still raised about ART, many infertile people have benefited from the development of modern ART. If the call for a moratorium [7] is intended to pause human germline-editing research (in addition to its clinical applications), the call appears to be too cautious about the socioethical implications of such research because many countries have already permitted human embryo research under relevant regulations [51] and have prohibited 7
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Opinion human germline gene modification for reproductive purposes [4]. If the calls [8,9] are intended to advocate that researchers are privileged to conduct human germlineediting research in a specific regulatory context, the outcomes of the research may, if not appropriately justified, lead the public towards absolute opposition to all in vitro experiments associated with human germline genome editing. If researchers believe that their research has sufficient justification to conduct human germline genome editing, they (excepting clinical applications) do not necessarily need to be subject to the moratorium when the research is to be conducted in countries where appropriate regulations are already in place. In such countries, researchers should fully discuss the socioethical implications of their research with the public. In this context, the reason(s) to conduct this research would preferably be medical – and not to enhance basic knowledge of the human germline [9]. Some researchers would explain their research from the perspective of future clinical applications, such as preventing the inheritance of genetic diseases before birth, as discussed in a recent movement on mitochondrial donation for mitochondrial diseases in the UK [6,11]. Other researchers may explain their research by focusing on understanding the molecular mechanisms underlying the onset of congenital anomalies. As a result, some research will be supported by society, while other projects will be discouraged or strictly regulated by newly established legislation. Although creating forums by experts, and holding international conferences, are both important [8], public dialogue will be vital to shape the socially-acceptable uses of germline genome editing (if any) and to avoid potential abuse of this technology. Acknowledgments The author thanks Motoko Araki for her help in the preparation of the figures. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant 26460586 (T.I.).
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