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Heritable human genome editing: Research progress, ethical considerations, and hurdles to clinical practice

      Summary

      Our genome at conception determines much of our health as an adult. Most human diseases have a heritable component and thus may be preventable through heritable genome editing. Preventing disease from the beginning of life before irreversible damage has occurred is an admirable goal, but the path to fruition remains unclear. Here, we review the significant scientific contributions to the field of human heritable genome editing, the unique ethical challenges that cannot be overlooked, and the hurdles that must be overcome prior to translating these technologies into clinical practice.

      Keywords

      Introduction

      The introduction of genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies generated tremendous enthusiasm as well as controversy within the medical and public communities. Heritable Human Genome Editing (HHGE) has the potential to treat or even eradicate genetic diseases. By addressing genetic disease before the defect is amplified through cell proliferation during development, HHGE may prove to be more effective than any other treatment being developed today, including somatic gene or drug therapy (Figure 1). Somatic gene therapies are limited in their ability to reverse damage that has already occurred and to reach the billions of cells needed to adequately treat the disease. The brief in vitro culture of a human embryo as routinely practiced in IVF clinics provides a readily accessible window for potential prevention of numerous conditions that later in life are difficult to manage, let alone cure. This hope provides the rationale for research, but not yet for therapy, as criteria of efficacy and safety have yet to be met. HHGE also raises difficult ethical and regulatory questions. Manipulations of the early embryo are highly consequential, both with regard to potential benefits but also with regard to risks. Other reviews have also discussed heritable genome editing, emphasizing its potential for understanding gene function in the early human embryo (
      • Lea R.A.
      • Niakan K.
      Human germline genome editing.
      ;
      • Rossant J.
      Gene editing in human development: ethical concerns and practical applications.
      ;
      • Plaza Reyes A.
      • Lanner F.
      Towards a CRISPR view of early human development: applications, limitations and ethical concerns of genome editing in human embryos.
      ). Here, we summarize the research on human heritable genome editing including its potential therapeutic value, ethical implications, and new risks that were identified in recent studies.
      Figure thumbnail gr1
      Figure 1Pathways to parenthood without disease
      Review of potential pathways to parenthood for couples with known heritable genetic disease who wish to avoid disease inheritance. Currently, couples may choose adoption, prenatal genetic diagnosis followed by selective pregnancy termination if affected, use of donor gametes, or preimplantation genetic testing for IVF-generated embryos. Mitochondrial replacement therapy and heritable human genome editing may provide a future pathway for couples wishing to avoid passing on a known genetic disease. Research in genome editing has targeted somatic cells, in utero fetal cells, embryos, gametes, as well as in vitro-derived gametes and haploid cells. Potential disease targets include single-gene, polygenic disease, disease risk alleles, aneuploidy, and mitochondrial disease.

      DNA double-strand breaks allow genome editing in human embryos

      Studies since the early 1980s have demonstrated the power of using DNA double-stand breaks (DSBs) for targeted genetic change, first in yeast and then in mammalian cells (reviewed by
      • Jasin M.
      • Rothstein R.
      Repair of strand breaks by homologous recombination.
      ). DNA DSBs may occur during the DNA replication process, after exposure to ionizing radiation or chemotherapy, or after experimental manipulation and are repaired mainly by one of two repair pathways: nonhomologous end joining (NHEJ) or homology directed repair (HDR). Based on the pioneering studies in model organisms, it became evident that genome editing through the targeted induction of a DSB may be an ideal approach for the correction of disease-causing mutations.
      A critical breakthrough was the discovery of DNA sequence specific nucleases amenable to design. Engineered nucleases can target a genetic sequence and create a DSB at a unique position. During the repair process, the original DNA sequence in the vicinity of the cut is altered, creating a new sequence. Several biological systems and types of nucleases such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) provide these capabilities for editing double-stranded DNA. Both TALENs and ZFNs consist of a combination of repetitive protein segments, each with DNA binding specificity of 1 or 3 nucleotides, to generate a protein with a DNA binding domain that is specific to a single site in the genome. However, it was the discovery of RNA-guided nucleases that made changes in the genome readily accessible and scalable for a wide range of applications. Shortly after the function of the RNA-guided endonuclease CRISPR-Cas9 was first described (
      • Gasiunas G.
      • Barrangou R.
      • Horvath P.
      • Siksnys V.
      Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.
      ;
      • Jinek M.
      • Chylinski K.
      • Fonfara I.
      • Hauer M.
      • Doudna J.A.
      • Charpentier E.
      A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
      ), it was adapted to modifying the genome of cultured mammalian cells (
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • Hsu P.D.
      • Wu X.
      • Jiang W.
      • Marraffini L.A.
      • Zhang F.
      Multiplex genome engineering using CRISPR/Cas systems.
      ;
      • Mali P.
      • Yang L.
      • Esvelt K.M.
      • Aach J.
      • Guell M.
      • DiCarlo J.E.
      • Norville J.E.
      • Church G.M.
      RNA-guided human genome engineering via Cas9.
      ) followed by human embryos (
      • Liang P.
      • Xu Y.
      • Zhang X.
      • Ding C.
      • Huang R.
      • Zhang Z.
      • Lv J.
      • Xie X.
      • Chen Y.
      • Li Y.
      • et al.
      CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
      ;
      • Kang X.
      • He W.
      • Huang Y.
      • Yu Q.
      • Chen Y.
      • Gao X.
      • Sun X.
      • Fan Y.
      Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.
      ;
      • Tang L.
      • Zeng Y.
      • Du H.
      • Gong M.
      • Peng J.
      • Zhang B.
      • Lei M.
      • Zhao F.
      • Wang W.
      • Li X.
      • Liu J.
      CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.
      ) and reviewed in
      • Lea R.A.
      • Niakan K.
      Human germline genome editing.
      .
      Initial CRISPR-Cas9-mediated studies in human embryos focused on rates of mutation correction, introduction of off-target edits, and mosaicism—multiple different genetic outcomes within the same embryo (
      • Liang P.
      • Xu Y.
      • Zhang X.
      • Ding C.
      • Huang R.
      • Zhang Z.
      • Lv J.
      • Xie X.
      • Chen Y.
      • Li Y.
      • et al.
      CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
      ;
      • Tang L.
      • Zeng Y.
      • Du H.
      • Gong M.
      • Peng J.
      • Zhang B.
      • Lei M.
      • Zhao F.
      • Wang W.
      • Li X.
      • Liu J.
      CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.
      ;
      • Kang X.
      • He W.
      • Huang Y.
      • Yu Q.
      • Chen Y.
      • Gao X.
      • Sun X.
      • Fan Y.
      Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.
      ) (Figure 2). The first such study made use of non-viable tripronuclear (3PN) embryos. While this experimental approach took advantage of embryos that would be clinically discarded, developmental outcomes and karyotypes are difficult to interpret and were hence not evaluated. After injecting 3PN zygotes with CRISPR-Cas9, Liang and colleagues found only 4 of 71 (5.6%) embryos contained the desired genetic change in hemoglobin β gene (HBB) (
      • Liang P.
      • Xu Y.
      • Zhang X.
      • Ding C.
      • Huang R.
      • Zhang Z.
      • Lv J.
      • Xie X.
      • Chen Y.
      • Li Y.
      • et al.
      CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
      ). The correctly edited embryos were mosaic and off-target mutations were common as the CRISPR-Cas9 complex was acting at two other sites of the genome. Kang and colleagues injected 126 3PN zygotes with Cas9 mRNA, guide RNA (gRNA), and correction template to modify the immune cell gene CCR5 (
      • Kang X.
      • He W.
      • Huang Y.
      • Yu Q.
      • Chen Y.
      • Gao X.
      • Sun X.
      • Fan Y.
      Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.
      ). Genetic analysis showed that 2 embryos were successfully modified with the intended 32 bp deletion. The use of two gRNAs to delete the same DNA segment without the need for HDR was successful in 4/26 embryos. Embryos with the intended genetic change were mosaic. Also using 3PN embryos, Tang et al. found integration of a homologous template in 2/30 (6.6%) zygotes at the G6PD locus, and 14% (2/14) showed homologous editing at the HBB locus (
      • Tang L.
      • Zeng Y.
      • Du H.
      • Gong M.
      • Peng J.
      • Zhang B.
      • Lei M.
      • Zhao F.
      • Wang W.
      • Li X.
      • Liu J.
      CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.
      ). Using diploid 2PN human embryos fertilized with mutant sperm,
      • Tang L.
      • Zeng Y.
      • Du H.
      • Gong M.
      • Peng J.
      • Zhang B.
      • Lei M.
      • Zhao F.
      • Wang W.
      • Li X.
      • Liu J.
      CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.
      showed that mutations in HBB and G6PD could be corrected. All told, however, the study was limited by the low number of embryos all of which proved to be mosaic.
      Figure thumbnail gr2
      Figure 2Efficiencies of genome-editing outcomes in human embryos
      Shown are human embryo genome editing outcomes with the stage of Cas9 injection specified. Genes that are not represented in all categories were either not studied for this aspect, or the number of embryos examined was deemed too low (2 or less). The endogenous repair template for HBB is the HBD locus, and the homologous chromosome is the repair template for EYS. *Number of embryos analyzed with 8 or more cells, from Figure 3a of
      • Fogarty N.M.E.
      • McCarthy A.
      • Snijders K.E.
      • Powell B.E.
      • Kubikova N.
      • Blakeley P.
      • Lea R.
      • Elder K.
      • Wamaitha S.E.
      • Kim D.
      • et al.
      Genome editing reveals a role for OCT4 in human embryogenesis.
      . 2-cell injections display the number of blastomeres for HDR, indels, and off-target activity instead of the number of embryos. Reference for HBB in 3PN embryos (
      • Liang P.
      • Xu Y.
      • Zhang X.
      • Ding C.
      • Huang R.
      • Zhang Z.
      • Lv J.
      • Xie X.
      • Chen Y.
      • Li Y.
      • et al.
      CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
      ), CCR5 (
      • Kang X.
      • He W.
      • Huang Y.
      • Yu Q.
      • Chen Y.
      • Gao X.
      • Sun X.
      • Fan Y.
      Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.
      ), G6PD and HBB in 2PN embryos (
      • Tang L.
      • Zeng Y.
      • Du H.
      • Gong M.
      • Peng J.
      • Zhang B.
      • Lei M.
      • Zhao F.
      • Wang W.
      • Li X.
      • Liu J.
      CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.
      ), MYBPC3 (
      • Ma H.
      • Marti-Gutierrez N.
      • Park S.W.
      • Wu J.
      • Lee Y.
      • Suzuki K.
      • Koski A.
      • Ji D.
      • Hayama T.
      • Ahmed R.
      • et al.
      Correction of a pathogenic gene mutation in human embryos.
      ), POU5F1 (
      • Alanis-Lobato G.
      • Zohren J.
      • Mccarthy A.
      • Fogarty N.M.E.
      • Kubikova N.
      • Hardman E.
      • Greco M.
      • Wells D.
      • Turner J.M.A.
      • Niakan K.K.
      Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos.
      ,
      • Fogarty N.M.E.
      • McCarthy A.
      • Snijders K.E.
      • Powell B.E.
      • Kubikova N.
      • Blakeley P.
      • Lea R.
      • Elder K.
      • Wamaitha S.E.
      • Kim D.
      • et al.
      Genome editing reveals a role for OCT4 in human embryogenesis.
      ), and EYS (
      • Zuccaro M.V.
      • Xu J.
      • Mitchell C.
      • Marin D.
      • Zimmerman R.
      • Rana B.
      • Weinstein E.
      • King R.T.
      • Palmerola K.L.
      • Smith M.E.
      • et al.
      Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.
      ). In Ma et al., the numbers of mutant embryos are inferred based on the frequency of mutant sperm from a heterozygous donor. This study also reported efficient HDR repair with the maternal chromosome as a template based on the absence of a disease-causing mutation at MYBPC3 (33% in zygotes [9/27] and 45% in MII oocytes [13/29]). The genetic nature of these embryos is not fully understood, as several outcomes, including chromosome loss, mitotic recombination, or more complex genetic change, could result in the absence of a detectable mutation. HDR, homology directed repair; 2PN, two pronuclear; 3PN, tripronuclear.
      Another study targeting MYBPC3, a mutation of which causes hypertrophic cardiomyopathy, also reported a low efficiency of template integration through HDR in 2PN embryos; only a single embryo out of 58 showed template integration in some of the cells (
      • Ma H.
      • Marti-Gutierrez N.
      • Park S.W.
      • Wu J.
      • Lee Y.
      • Suzuki K.
      • Koski A.
      • Ji D.
      • Hayama T.
      • Ahmed R.
      • et al.
      Correction of a pathogenic gene mutation in human embryos.
      ). As the sperm donor used was heterozygous, an estimated 29 embryos were generated with mutant sperm, resulting in an HDR efficiency using the template of ∼3%.
      For HDR to be therapeutically relevant for the precise repair of disease-causing mutations in the human embryos, the efficiency of editing will need to be increased. Low efficiency of HDR may be due to the lack of control over when DSBs occur and in which cell-cycle phase. HDR is more active in the S and G2 phases than in G1, though this assertion has not been directly tested in human embryos. Furthermore, the timing of the cell-cycle phases and of the kinetics of Cas9 cleavage in the human embryo have not yet been determined. Timing of microinjection at the G2 phase of the cell cycle has proved useful in promoting HDR in the mouse embryo (
      • Gu B.
      • Posfai E.
      • Rossant J.
      Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.
      ). According to
      • Gu B.
      • Posfai E.
      • Rossant J.
      Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.
      , the efficiency of targeted integration could be further increased to 95% by tethering a biotinylated DNA repair template to Cas9, which was modified by fusion to monomeric avidin. This approach has not yet been tested in human embryos but appears promising. In embryonic stem cells as well as somatic cells, the efficiency of homologous recombination can be increased ∼2-fold through interference with the function of 53BP1 (
      • Nambiar T.S.
      • Billon P.
      • Diedenhofen G.
      • Hayward S.B.
      • Taglialatela A.
      • Cai K.
      • Huang J.W.
      • Leuzzi G.
      • Cuella-Martin R.
      • Palacios A.
      • et al.
      Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant.
      ;
      • Canny M.D.
      • Moatti N.
      • Wan L.C.K.
      • Fradet-Turcotte A.
      • Krasner D.
      • Mateos-Gomez P.A.
      • Zimmermann M.
      • Orthwein A.
      • Juang Y.C.
      • Zhang W.
      • et al.
      Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency.
      ). 53BP1 inhibits DSB repair by HDR and promotes NHEJ (
      • Bunting S.F.
      • Callén E.
      • Wong N.
      • Chen H.T.
      • Polato F.
      • Gunn A.
      • Bothmer A.
      • Feldhahn N.
      • Fernandez-Capetillo O.
      • Cao L.
      • et al.
      53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.
      ), and thus, through its deletion, more DSBs are repaired by HDR, though the effect is modest. An alternative approach is to increase the expression of factors involved in HDR. Injection of mouse zygotes with Rad51, a protein involved in the search for a homologous repair template, appears to increase HDR efficiency in mouse embryos (
      • Wilde J.J.
      • Aida T.
      • Wienisch M.
      • Zhang Q.
      • Qi P.
      • Feng G.
      Efficient Zygotic Genome Editing via RAD51-Enhanced Interhomolog Repair.
      ). However, reliance on Rad51 could also result in detrimental genetic changes including translocations due to increased recombination between homologous sequences in the human genome (
      • Richardson C.
      • Stark J.M.
      • Ommundsen M.
      • Jasin M.
      Rad51 overexpression promotes alternative double-strand break repair pathways and genome instability.
      ). Neither approach has thus far been tested in human embryos.
      Most groups have focused on using HDR to introduce programmed edits into the human germline genome (
      • Liang P.
      • Xu Y.
      • Zhang X.
      • Ding C.
      • Huang R.
      • Zhang Z.
      • Lv J.
      • Xie X.
      • Chen Y.
      • Li Y.
      • et al.
      CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.
      ;
      • Kang X.
      • He W.
      • Huang Y.
      • Yu Q.
      • Chen Y.
      • Gao X.
      • Sun X.
      • Fan Y.
      Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.
      ;
      • Tang L.
      • Zeng Y.
      • Du H.
      • Gong M.
      • Peng J.
      • Zhang B.
      • Lei M.
      • Zhao F.
      • Wang W.
      • Li X.
      • Liu J.
      CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.
      ;
      • Ma H.
      • Marti-Gutierrez N.
      • Park S.W.
      • Wu J.
      • Lee Y.
      • Suzuki K.
      • Koski A.
      • Ji D.
      • Hayama T.
      • Ahmed R.
      • et al.
      Correction of a pathogenic gene mutation in human embryos.
      ). However, NHEJ repair may also be used to restore or disrupt gene function. The outcomes are generally novel alleles that have no precedent in the human population. For instance, recurrent end-joining events restore the reading frame of a mutation at the EYS locus but results in alleles with an altered amino acid sequence relative to the wild type (
      • Zuccaro M.V.
      • Xu J.
      • Mitchell C.
      • Marin D.
      • Zimmerman R.
      • Rana B.
      • Weinstein E.
      • King R.T.
      • Palmerola K.L.
      • Smith M.E.
      • et al.
      Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.
      ). The generation of novel alleles may be very useful in a research context, such as to study the function of a gene product in early embryonic development. This experimental approach has been used to identify a requirement of POU5F1 for normal blastocyst development (
      • Fogarty N.M.E.
      • McCarthy A.
      • Snijders K.E.
      • Powell B.E.
      • Kubikova N.
      • Blakeley P.
      • Lea R.
      • Elder K.
      • Wamaitha S.E.
      • Kim D.
      • et al.
      Genome editing reveals a role for OCT4 in human embryogenesis.
      ). However, in the context of reproduction, functional testing of a novel allele in a human being is not possible, or acceptable, and therefore the effect on the health of a person cannot be known. A recent report by the Royal Society and the National Academies of Sciences, Engineering, and Medicine specifically called for only the introduction of common variants in the relevant population to correct a mutation (
      NAS, National Academy of Medicine, National Academy of Sciences and the Royal Society
      Heritable Human Genome Editing.
      ). Thus, HDR is preferable to NHEJ as a path to germline gene correction. However, low efficiency of HDR was found in all studies; in aggregate, less than 10% of cells showed the intended modification. The formation of indels predominates about 10-fold (Figure 2).

      Adverse consequences of DNA breaks in human embryos

      In addition to small indels and precise repair by homologous recombination, Cas9-induced DSBs can also result in more extensive genetic changes. In mouse embryos, Cas9 gave rise to deletions of several hundred base pairs in almost half of the embryos (
      • Adikusuma F.
      • Piltz S.
      • Corbett M.A.
      • Turvey M.
      • McColl S.R.
      • Helbig K.J.
      • Beard M.R.
      • Hughes J.
      • Pomerantz R.T.
      • Thomas P.Q.
      Large deletions induced by Cas9 cleavage.
      ). Such deletions are also common in mouse embryonic stem cells (
      • Kosicki M.
      • Tomberg K.
      • Bradley A.
      Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.
      ). Kosicki and colleagues found that more than 20% of the targeted alleles in mouse embryonic stem cells contained large deletions >250 bp extending up to 6 kb away from the CRISPR cut site. Mutations also included complex genomic rearrangements at the targeted sites. In contrast, Ma and colleagues noted a lack of large deletions by performing long-range PCR and SNP analysis in human embryos (
      • Ma H.
      • Marti-Gutierrez N.
      • Park S.W.
      • Wu J.
      • Hayama T.
      • Darby H.
      • Van Dyken C.
      • Li Y.
      • Koski A.
      • Liang D.
      • et al.
      Ma et al. reply.
      ). Large deletions were similarly missing in another study targeting the EYS locus (
      • Zuccaro M.V.
      • Xu J.
      • Mitchell C.
      • Marin D.
      • Zimmerman R.
      • Rana B.
      • Weinstein E.
      • King R.T.
      • Palmerola K.L.
      • Smith M.E.
      • et al.
      Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.
      ). Fogarty and colleagues found deletions of up to ∼28 bp (
      • Fogarty N.M.E.
      • McCarthy A.
      • Snijders K.E.
      • Powell B.E.
      • Kubikova N.
      • Blakeley P.
      • Lea R.
      • Elder K.
      • Wamaitha S.E.
      • Kim D.
      • et al.
      Genome editing reveals a role for OCT4 in human embryogenesis.
      ). Though this observation does not exclude the possibility of large deletions, human embryos do not appear to incur deletions of several hundred base pairs at the frequency seen in mice, thereby pointing to species-specific differences in DSB repair.
      Surprisingly, Cas9 resulted in frequent chromosomal changes in human embryos. Zuccaro and colleagues targeted a blindness-causing mutation in the EYS gene with CRISPR-Cas9 (
      • Zuccaro M.V.
      • Xu J.
      • Mitchell C.
      • Marin D.
      • Zimmerman R.
      • Rana B.
      • Weinstein E.
      • King R.T.
      • Palmerola K.L.
      • Smith M.E.
      • et al.
      Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.
      ). DNA breaks in approximately half of the embryos injected remained unrepaired, resulting in the loss of a chromosome arm or a whole chromosome. Similarly,
      • Fogarty N.M.E.
      • McCarthy A.
      • Snijders K.E.
      • Powell B.E.
      • Kubikova N.
      • Blakeley P.
      • Lea R.
      • Elder K.
      • Wamaitha S.E.
      • Kim D.
      • et al.
      Genome editing reveals a role for OCT4 in human embryogenesis.
      used CRISPR-Cas9 to create mutations in the POU5F1 gene for embryo developmental studies and also found chromosomal changes (
      • Alanis-Lobato G.
      • Zohren J.
      • Mccarthy A.
      • Fogarty N.M.E.
      • Kubikova N.
      • Hardman E.
      • Greco M.
      • Wells D.
      • Turner J.M.A.
      • Niakan K.K.
      Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos.
      ). The multiple possible outcomes associated with the generation of a DSB present daunting challenges. Chromosomal changes as a consequence of Cas9 cleavage can also occur in human differentiated cells, though at about 10-fold lower frequency than in human embryos (
      • Leibowitz M.L.
      • Papathanasiou S.
      • Doerfler P.A.
      • Blaine L.J.
      • Yao Y.
      • Zhang C.-Z.
      • Weiss M.J.
      • Pellman D.
      Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.
      ), pointing to differences in DNA repair in embryos and somatic cells. A large body of work will be needed to better understand the endogenous embryo repair machinery and increase the frequency of desired repair outcomes.
      A primary limitation of all gene-editing approaches is the ability to prevent mosaicism. By restricting the activity of gene editors to the first cell cycle, and potentially in advance of the replication of the targeted locus, mosaicism could potentially be avoided. The discovery of anti-CRISPR proteins (
      • Pawluk A.
      • Davidson A.R.
      • Maxwell K.L.
      Anti-CRISPR: discovery, mechanism and function.
      ) may well enable such temporal control through timed injection of the anti-CRISPR protein relative to Cas9. Interestingly, Cas9 activity at off-target sites appears to occur with a delay relative to the on-target site in human embryos. While the majority of on-target sites were modified within the first cell cycle of embryo development, most off-target genetic change occurred in later cell cycles and were mosaic (
      • Zuccaro M.V.
      • Xu J.
      • Mitchell C.
      • Marin D.
      • Zimmerman R.
      • Rana B.
      • Weinstein E.
      • King R.T.
      • Palmerola K.L.
      • Smith M.E.
      • et al.
      Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.
      ). In somatic cells, delayed addition of the anti-Crispr protein AcrIIA4 inhibits off-target cleavage while still allowing on target activity (
      • Shin J.
      • Jiang F.
      • Liu J.-J.
      • Bray N.L.
      • Rauch B.J.
      • Baik S.H.
      • Nogales E.
      • Bondy-Denomy J.
      • Corn J.E.
      • Doudna J.A.
      Disabling Cas9 by an anti-CRISPR DNA mimic.
      ). Anti-Crispr proteins have promise to reduce on-target and off-target mosaicism but have not as yet been tested in human embryos.

      Base and prime editing in human embryos

      Recently developed editing systems such as base and prime editing do not use DSBs and thus may avoid some of the aforementioned undesirable repair outcomes. In base editing, a catalytically inactive Cas9 serves to guide a deaminase to a specific site in the genome to mediate site-specific nucleotide to nucleotide conversions (
      • Komor A.C.
      • Kim Y.B.
      • Packer M.S.
      • Zuris J.A.
      • Liu D.R.
      Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
      ). Base editing has been tested in human embryos (
      • Zhou C.
      • Zhang M.
      • Wei Y.
      • Sun Y.
      • Sun Y.
      • Pan H.
      • Yao N.
      • Zhong W.
      • Li Y.
      • Li W.
      • et al.
      Highly efficient base editing in human tripronuclear zygotes.
      ;
      • Zhang M.
      • Zhou C.
      • Wei Y.
      • Xu C.
      • Pan H.
      • Ying W.
      • Sun Y.
      • Sun Y.
      • Xiao Q.
      • Yao N.
      • et al.
      Human cleaving embryos enable robust homozygotic nucleotide substitutions by base.
      ;
      • Li G.
      • Liu Y.
      • Zeng Y.
      • Li J.
      • Wang L.
      • Yang G.
      • Chen D.
      • Shang X.
      • Chen J.
      • Huang X.
      • Liu J.
      Highly efficient and precise base editing in discarded human tripronuclear embryos.
      ,
      • Zeng Y.
      • Li J.
      • Li G.
      • Huang S.
      • Yu W.
      • Zhang Y.
      • Chen D.
      • Chen J.
      • Liu J.
      • Huang X.
      Correction of the Marfan Syndrome Pathogenic FBN1 Mutation by Base Editing in Human Cells and Heterozygous Embryos.
      ) and was found to be highly efficient: editing efficiency was higher than 50% in most cases. Indels occurred at a lower frequency than after Cas9 cleavage and may be due to a response of the endogenous repair mechanisms to the deaminated base. Studies also reported mosaicism and base changes at sites flanking the targeted base. Base editors with a more limited editing window and less bystander effects have been developed (
      • Kim Y.B.
      • Komor A.C.
      • Levy J.M.
      • Packer M.S.
      • Zhao K.T.
      • Liu D.R.
      Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.
      ) but have not as of yet been tested in human embryos.
      While more efficient than HDR, base editing can only correct four out of 12 nucleotide substitutions and cannot repair genetic changes such as indels. About 60% of disease-causing mutations may be corrected using base editing (
      • Rees H.A.
      • Liu D.R.
      Base editing: precision chemistry on the genome and transcriptome of living cells.
      ). In 2019, Anzalone and colleagues described the prime editing method to overcome these limitations. Prime editing uses a Cas9 nickase for the recruitment of a reverse transcriptase to the target site and the introduction of a break in just one of the two DNA strands. The attendant gRNA is modified to include an RNA template for the repair of the mutation by copying it through reverse transcription into the single-stranded nick (
      • Anzalone A.V.
      • Randolph P.B.
      • Davis J.R.
      • Sousa A.A.
      • Koblan L.W.
      • Levy J.M.
      • Chen P.J.
      • Wilson C.
      • Newby G.A.
      • Raguram A.
      • Liu D.R.
      Search-and-replace genome editing without double-strand breaks or donor DNA.
      ). Prime editing also allows for the repair of a much wider spectrum of mutations without the need for a donor DNA template or the generation of a DSB. Using prime editing to correct the genes responsible for sickle cell disease and Tay Sachs, Liu’s group reported a higher or similar efficiency and lower off-target editing compared to CRISPR-triggered HDR (
      • Anzalone A.V.
      • Randolph P.B.
      • Davis J.R.
      • Sousa A.A.
      • Koblan L.W.
      • Levy J.M.
      • Chen P.J.
      • Wilson C.
      • Newby G.A.
      • Raguram A.
      • Liu D.R.
      Search-and-replace genome editing without double-strand breaks or donor DNA.
      ). While the key steps of gene editing are performed by exogenously engineered enzymes, the endogenous DNA repair machinery is still required to seal the nick induced in the DNA. Thus, the outcomes of prime editing may be cell type and species dependent. It is therefore important to study the consequences of this approach directly in the relevant cell type. In mouse embryos, intended edits could be introduced in 10%–50% of the embryos at different loci (
      • Aida T.
      • Wilde J.J.
      • Yang L.
      • Hou Y.
      • Li M.
      • Xu D.
      • Lin J.
      • Qi P.
      • Lu Z.
      • Feng G.
      Prime editing primarily induces undesired outcomes in mice.
      ;
      • Liu Y.
      • Li X.
      • He S.
      • Huang S.
      • Li C.
      • Chen Y.
      • Liu Z.
      • Huang X.
      • Wang X.
      Efficient generation of mouse models with the prime editing system.
      ). However, in addition to the intended genetic change, indels were also observed in up to 60% of the embryos, which proved mosaic for both intended and unintended genetic change. Prime editing has yet to be tested in human embryos, and there is insufficient understanding of endogenous DNA repair pathways in the human embryo to anticipate its associated outcomes.

      Mitochondrial replacement therapy

      In addition to nuclear DNA mutations, heritable genetic disease can also be caused by mutations in mitochondrial DNA. Mitochondrial disorders are among the most common inherited metabolic diseases and can be debilitating or fatal at an early age. Given the lack of effective pharmacologic agents for the treatment of mitochondrial DNA disorders, current treatment is largely supportive. Forms of prevention have focused on preimplantation genetic diagnosis or mitochondrial replacement. Mitochondrial replacement entails the replacement of the mutated mitochondrial DNA of the oocyte with a healthy mitochondrial genome from a donated oocyte (
      • Herbert M.
      • Turnbull D.
      Progress in mitochondrial replacement therapies.
      ). Nuclear content of a maternal oocyte is transferred to a normal “enucleated’ oocyte from a donor female prior to implantation. The offspring will have all the nuclear genetic components of the parents but mitochondrial DNA from a female donor. Mitochondrial replacement therapy has been approved for use in the United Kingdom strictly to prevent genetic disease, and a clinical trial is ongoing. In 2016, mitochondrial replacement therapy was used to avoid transmitting the hereditary disease, Leigh syndrome, resulting in a child with predominantly normal mitochondrial DNA (
      • Zhang J.
      • Liu H.
      • Luo S.
      • Lu Z.
      • Chávez-Badiola A.
      • Liu Z.
      • Yang M.
      • Merhi Z.
      • Silber S.J.
      • Munné S.
      • et al.
      Live birth derived from oocyte spindle transfer to prevent mitochondrial disease.
      ).
      Within an oocyte, thousands of mitochondrial DNA molecules exist. Mutant and wild-type mitochondrial DNA may co-exist and only result in disease if the mutant mitochondrial DNA exceeds a certain threshold. The specific cleavage of mutant mitochondria through TALENs to reduce the percentage of mutant mitochondrial DNA may be an alternative approach to treating mitochondrial disease (
      • Reddy P.
      • Ocampo A.
      • Suzuki K.
      • Luo J.
      • Bacman S.R.
      • Williams S.L.
      • Sugawara A.
      • Okamura D.
      • Tsunekawa Y.
      • Wu J.
      • et al.
      Selective elimination of mitochondrial mutations in the germline by genome editing.
      ). Only recently has it become possible to edit the mitochondrial genome using TALEN nucleases fused to a deaminase that can act on double-stranded DNA (
      • Mok B.Y.
      • de Moraes M.H.
      • Zeng J.
      • Bosch D.E.
      • Kotrys A.V.
      • Raguram A.
      • Hsu F.
      • Radey M.C.
      • Peterson S.B.
      • Mootha V.K.
      • et al.
      A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.
      ). This latest base editor adds yet another possible tool to the prevention of mitochondrial DNA disease in the context of human reproduction. Neither approach has been tested in human embryos.
      An important distinction between mitochondrial replacement and the use of TALENs for cleavage or editing is that mitochondrial replacement does not involve any direct change to the genome itself. Rather, it is a manipulation to alter the pattern of mitochondrial DNA inheritance and provides no path to introducing genetic variants that do not already exist in the human population. Thus, some of the concerns relating to heritable genome editing do not apply to this technology.

      Editing of in vitro-derived gametes and embryos

      Spermatogonial stem cells

      Cultured cells provide several practical advantages for gene editing over embryos. They allow for a larger number of modifications, a comprehensive genetic analysis prior to generating an embryo, and the ability to avoid mosaicism. Several different cell types have reproductive potential. In men, germline cells continue to proliferate throughout adulthood as spermatogonial stem cells and may provide a path to generating edited mature sperm.
      • Wu Y.
      • Zhou H.
      • Fan X.
      • Zhang Y.
      • Zhang M.
      • Wang Y.
      • Xie Z.
      • Bai M.
      • Yin Q.
      • Liang D.
      • et al.
      Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells.
      reported the editing of mouse spermatogonial stem cells followed by testicular transplantation, resulting in the repair of a cataract-causing mutation. Fertilization using spermatids derived from these edited spermatogonial stem cells gave rise to offspring with the corrected phenotype at 100% efficiency. Gene editing of spermatogonial stem cells, perhaps even in vivo, might provide a path to efficient gene editing of the paternal genome while also avoiding mosaicism. This approach could, for instance, be used to restore spermatogenesis due to mutations in genes required for sperm maturation and prevent offspring from inheriting these same mutations.

      Induced pluripotent stem cells

      In vitro-derived gametes induced from pluripotent cells are also an active area of research. Pluripotent stem cells are frequently used as a target for genome editing in the context of human disease modeling. For example,
      • Schwank G.
      • Koo B.K.
      • Sasselli V.
      • Dekkers J.F.
      • Heo I.
      • Demircan T.
      • Sasaki N.
      • Boymans S.
      • Cuppen E.
      • van der Ent C.K.
      • et al.
      Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.
      used CRISPR-Cas9 to successfully correct cystic fibrosis mutations in induced pluripotent stem cells derived from cystic fibrosis patients. Pluripotent cells can give rise to all cell types of the body including germ cells. When induced from somatic cells, they are diploid and need to undergo meiosis for use in reproduction.
      • Hayashi K.
      • Ohta H.
      • Kurimoto K.
      • Aramaki S.
      • Saitou M.
      Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.
      reported mouse primordial germ cell-like cells derived from male pluripotent stem cells that have been transplanted into the seminiferous tubules of germ cell-ablated mice and yielded functional sperm. Hayashi also showed mouse primordial germ cell-like cells derived from female embryonic stem cells and induced pluripotent stem cells developed into fully grown oocytes that contributed to healthy offspring (
      • Hayashi K.
      • Ogushi S.
      • Kurimoto K.
      • Shimamoto S.
      • Ohta H.
      • Saitou M.
      Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice.
      ). More recently, Hayashi’s laboratory identified transcription factors that allow conversion of mouse pluripotent stem cells to oocyte-like cells that proved fertilization competent, although they failed to undergo normal meiosis and give rise to embryos (
      • Hamazaki N.
      • Kyogoku H.
      • Araki H.
      • Miura F.
      • Horikawa C.
      • Hamada N.
      • Shimamoto S.
      • Hikabe O.
      • Nakashima K.
      • Kitajima T.S.
      • et al.
      Reconstitution of the oocyte transcriptional network with transcription factors.
      ). Though primordial germ cell-like cells have been made from human pluripotent stem cells (
      • Yamashiro C.
      • Sasaki K.
      • Yabuta Y.
      • Kojima Y.
      • Nakamura T.
      • Okamoto I.
      • Yokobayashi S.
      • Murase Y.
      • Ishikura Y.
      • Shirane K.
      • et al.
      Generation of human oogonia from induced pluripotent stem cells in vitro.
      ), no mature gametes have been reported to date.

      Haploid human stem cells

      Further progress toward reproduction has been made in haploid human stem cells (Figure 3). Haploid human pluripotent stem cells are derived from either parthenogenetically activated oocytes that develop without fertilization (
      • Sagi I.
      • Chia G.
      • Golan-Lev T.
      • Peretz M.
      • Weissbein U.
      • Sui L.
      • Sauer M.V.
      • Yanuka O.
      • Egli D.
      • Benvenisty N.
      Derivation and differentiation of haploid human embryonic stem cells.
      ) or from sperm injected into enucleated oocytes (
      • Zhang X.M.
      • Wu K.
      • Zheng Y.
      • Zhao H.
      • Gao J.
      • Hou Z.
      • Zhang M.
      • Liao J.
      • Zhang J.
      • Gao Y.
      • et al.
      In vitro expansion of human sperm through nuclear transfer.
      ). Haploid pluripotent stem cells have the chromosomal equivalent of a gamete, containing only 23 chromosomes. Remarkably, haploid stem cells derived from sperm can act like sperm fertilize a human oocyte and allow development to the blastocyst stage (
      • Zhang X.M.
      • Wu K.
      • Zheng Y.
      • Zhao H.
      • Gao J.
      • Hou Z.
      • Zhang M.
      • Liao J.
      • Zhang J.
      • Gao Y.
      • et al.
      In vitro expansion of human sperm through nuclear transfer.
      ). Imprinting patterns of these embryos are indistinguishable from IVF controls, and gene-expression patterns were very similar, though a small number of genes of unknown significance were differentially expressed. Developmental efficiency to the blastocyst stage was lower than in ICSI control embryos; of 130 oocytes injected with paternal haploid cells, only 5 formed euploid blastocysts. Pregnancy was not attempted with these 5 euploid blastocysts.
      Figure thumbnail gr3
      Figure 3Editing haploid human pluripotent stem cells
      Shown is a proposed pathway for reproduction with genome-edited paternal haploid stem cells: (1) The maternal genome is removed from the oocyte followed by sperm injection. The resultant haploid embryo is cultured to the blastocyst stage where the inner cell mass is used to generate a haploid pluripotent embryonic stem cell (ESC) line. Only X sperm give rise to ESCs as monosomy Y is lethal. (2) One or multiple edits can be performed with CRISPR/Cas9 or prime editing in ESCs followed by clonal expansion. Whole genome sequencing can identify genetic mutations including novel, culture-induced genetic, and/or epigenetic changes. (3) Haploid cells spontaneously convert to diploid cells at a rate of 1–5% per day, requiring selection of haploid cells through sorting for low (haploid) DNA content (
      • Sagi I.
      • Chia G.
      • Golan-Lev T.
      • Peretz M.
      • Weissbein U.
      • Sui L.
      • Sauer M.V.
      • Yanuka O.
      • Egli D.
      • Benvenisty N.
      Derivation and differentiation of haploid human embryonic stem cells.
      ). (4) Instead of fertilization by sperm, a single haploid ESC with an edited paternal genome is injected into a nucleated oocyte. Because ESCs contain mitochondrial DNA from the oocyte donor used for derivation, a match of mitochondria DNA genotypes might be needed to ensure stability of the mitochondria DNA genotype. (5) The resulting diploid embryo is cultured and biopsied for genome sequencing and epigenetic characterization. Maternal and paternal genomes in the embryo result in a diploid, 46 XX karyotype. The last step, to establish a pregnancy for the birth of a child, has not been attempted. For use of a maternal haploid ESC, its edited genome would replace the genome of the oocyte, and would be fertilized by sperm, allowing both female and male offspring (not illustrated). Below: tools for genome editing. Cas9 induces a double-stranded break, which can be repaired with a homologous template or through end joining. Cleavage between regions of microhomology (red nucleotides) can result in predictable outcomes. In contrast, prime editing, as well as base editing (not illustrated), involve a single strand DNA cut.
      Haploid cells can be clonally expanded thereby allowing extensive genetic modification, selection, and detailed genetic and epigenetic analysis. Editing in haploid stem cells is efficient (
      • Safier L.Z.
      • Zuccaro M.V.
      • Egli D.
      Efficient SNP editing in haploid human pluripotent stem cells.
      ), and their genomes have been modified at scale in the context of genetic screens (
      • Yilmaz A.
      • Braverman-Gross C.
      • Bialer-Tsypin A.
      • Peretz M.
      • Benvenisty N.
      Mapping Gene Circuits Essential for Germ Layer Differentiation via Loss-of-Function Screens in Haploid Human Embryonic Stem Cells.
      ). Sequential editing or multiplexing may allow one to eliminate damaging variants from the human genome. The estimated number of mutations disrupting protein coding genes in the human genome is ∼100 (
      • MacArthur D.G.
      • Balasubramanian S.
      • Frankish A.
      • Huang N.
      • Morris J.
      • Walter K.
      • Jostins L.
      • Habegger L.
      • Pickrell J.K.
      • Montgomery S.B.
      • et al.
      1000 Genomes Project Consortium
      A systematic survey of loss-of-function variants in human protein-coding genes.
      ). However, there is not yet sufficient understanding of the health impact of these mutations and whether or not they might contribute to normal phenotypic variation. Such information may ultimately be gained through a combination of genome sequencing, and molecular and functional studies in cellular models of human disease as well as in animal models. These efforts are ongoing worldwide to understand mechanisms of human disease for the development of adult therapies. Though the motivation is different and independent of germline genome therapy, these efforts will also inform which and how many variants might be meaningful to target in the germline.
      While the correction of numerous disease-causing variants in cultured cells may be an attractive concept, routine culture will also introduce novel mutations as well as epigenetic changes, which could affect the health of the resulting embryo. By some measurements, mutation rates in human pluripotent stem cells are about 3–4 base substitutions per genome and population doubling, or within about a day of culture (
      • Kuijk E.
      • Jager M.
      • van der Roest B.
      • Locati M.D.
      • Van Hoeck A.
      • Korzelius J.
      • Janssen R.
      • Besselink N.
      • Boymans S.
      • van Boxtel R.
      • Cuppen E.
      The mutational impact of culturing human pluripotent and adult stem cells.
      ). Culture-induced genetic or epigenetic change will be clonally inherited and remain of unknown functional significance until a human being is made. Compared to the use of sperm and oocytes, reproduction from cultured pluripotent stem cells adds a sequence of cellular manipulations with high risks. This is particularly true for induced pluripotent stem cells derived from somatic cells, which contain mutations of somatic origin as well as from in vitro reprogramming (
      • Gore A.
      • Li Z.
      • Fung H.L.
      • Young J.E.
      • Agarwal S.
      • Antosiewicz-Bourget J.
      • Canto I.
      • Giorgetti A.
      • Israel M.A.
      • Kiskinis E.
      • et al.
      Somatic coding mutations in human induced pluripotent stem cells.
      ;
      • Bhutani K.
      • Nazor K.L.
      • Williams R.
      • Tran H.
      • Dai H.
      • Džakula Ž.
      • Cho E.H.
      • Pang A.W.C.
      • Rao M.
      • Cao H.
      • et al.
      Whole-genome mutational burden analysis of three pluripotency induction methods.
      ;
      • Rouhani F.J.
      • Nik-Zainal S.
      • Wuster A.
      • Li Y.
      • Conte N.
      • Koike-Yusa H.
      • Kumasaka N.
      • Vallier L.
      • Yusa K.
      • Bradley A.
      Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.
      ;
      • Yoshihara M.
      • Araki R.
      • Kasama Y.
      • Sunayama M.
      • Abe M.
      • Nishida K.
      • Kawaji H.
      • Hayashizaki Y.
      • Murakawa Y.
      Hotspots of De Novo Point Mutations in Induced Pluripotent Stem Cells.
      ). Thus, this approach, in most instances, will be inconsistent with the above-stated goal of avoiding the introduction of novel alleles. Further considerations under which circumstances such risk might be justified will need to be made.

      Ethical considerations of heritable human genome editing

      With the introduction of efficient genome editing tools such as CRISPR-Cas9, the plausibility of safely editing the genome of the human germline is currently the subject of many academic, industry, and policy discussions. In contrast to gene editing in somatic cells, gene editing in human gametes or embryos to permanently modify the germline raises significant ethical concerns. Shortly before the 2018 International Human Genome Editing Summit, reports emerged that two girls were born after germline genome editing to prevent the expression of the HIV receptor CCR5 (
      • Cyranoski D.
      • Ledford H.
      Genome-edited baby claim provokes international outcry.
      ). This announcement was met with strong statements condemning the practice in general, the lack of a sound medical basis, the lack of safety assessments, the inadequacy of the informed-consent documents signed by the prospective parents, and the lack of public discussion and input regarding the personal and societal consequences of HHGE (
      • Savulescu J.
      • Singer P.
      An ethical pathway for gene editing.
      ).
      Many called for a moratorium on clinical HHGE (
      • Lander E.S.
      • Baylis F.
      • Zhang F.
      • Charpentier E.
      • Berg P.
      • Bourgain C.
      • Friedrich B.
      • Joung J.K.
      • Li J.
      • Liu D.
      • et al.
      Adopt a moratorium on heritable genome editing.
      ) and the ethical debate on HHGE continues to intensify. Because it is associated with human reproduction, HHGE often evokes spiritual, religious, or deeply personal issues for many. The following ethical discussion is not meant to be exhaustive but instead focuses on key issues surrounding HHGE as it relates to the four core bioethical principles: beneficence, non-maleficence, autonomy, and justice (Figure 4).
      Figure thumbnail gr4
      Figure 4Ethical considerations of heritable human genome editing
      The four principles of biomedical ethics (beneficence, non-maleficence, autonomy, and justice) are used as framework to describe the ethical concerns surrounding human heritable genome editing. (A) Beneficence: Genome editing has the potential to treat or even eradicate heritable disease. (B) Autonomy: Parents have right to make their own reproductive decisions which will affect offspring and subsequent generations. (C) Non-maleficence: Unintended outcomes of CRISPR/Cas9 can have dire consequences. Some worry germline editing is a slippery slope that can lead to “designer babies” and affect genetic and phenotypic diversity in human reproduction. (D) Justice: Access to assisted reproductive technologies is limited to individuals and countries who can afford it (figured adapted from (
      • DeWeerdt S.
      How much should having a baby cost?.
      ).

      Beneficence: Therapeutic benefits

      With over 10,000 known monogenic diseases, heritable diseases collectively affect roughly 5%–7% of the human population (
      • Kofler N.
      • Kraschel K.L.
      Treatment of heritable diseases using CRISPR: Hopes, fears, and reality.
      ). Millions more are affected by genetic variants that increase disease risk to common disorders such as diabetes, obesity, or cardiovascular disease. The correction of mutations in the germline would allow patients to create embryos free of disease-causing mutations. The disease gene would no longer be passed on to subsequent generations and over multiple generations. This alone could reduce disease prevalence and even eliminate selected heritable diseases. Some argue that the medical need for HHGE is so compelling that proceeding with this use of genome editing is a moral imperative (
      • Gyngell C.
      • Bowman-Smart H.
      • Savulescu J.
      Moral reasons to edit the human genome: picking up from the Nuffield report.
      ) and that doing so would help to “lighten the burden of human existence” (
      • Harris J.
      Germline Modification and the Burden of Human Existence.
      ).
      Some may argue that IVF with preimplantation genetic testing for monogenic disease (PGT-M) already allows couples to have genetically related children without the risk of inheriting a known familial disorder (
      NAS, National Academy of Medicine, National Academy of Sciences and the Royal Society
      Heritable Human Genome Editing.
      ). Patients with a genetic disorder can use PGT-M to screen IVF embryos for the disease-causing gene of interest and transfer only disease-free embryos. Few scenarios exist in which couples would not be eligible for PGT-M and would necessitate the use of HHGE. For instance, if a parent is homozygous for an autosomal dominant disorder, every embryo will inherit at least one copy of the causative allele and will thus be affected. If both parents are homozygous for a recessive disorder such as cystic fibrosis, all embryos will be homozygous. Given the rarity of these scenarios, analysis of prevalence data of common genetic disorders suggests that the clinical need for HHGE for cases that are not amenable to PGT-M is exceedingly small (
      • Viotti M.
      • Victor A.R.
      • Griffin D.K.
      • Groob J.S.
      • Brake A.J.
      • Zouves C.G.
      • Barnes F.L.
      Estimating Demand for Germline Genome Editing: An In Vitro Fertilization Clinic Perspective.
      ). An analysis by
      • Viotti M.
      • Victor A.R.
      • Griffin D.K.
      • Groob J.S.
      • Brake A.J.
      • Zouves C.G.
      • Barnes F.L.
      Estimating Demand for Germline Genome Editing: An In Vitro Fertilization Clinic Perspective.
      estimated that if all of the patients who are ineligible for PGT-M opted for HHGE, HHGE would benefit at most 100 births per year in the United States.
      Preimplantation genetic testing, however, is not equally effective for all couples. Even without PGT-M, a limited number of oocytes retrieved during IVF will fertilize and result in an embryo for transfer. IVF success rates decline significantly with age with only an estimated 12% of oocytes retrieved result in live birth (
      • De Rycke M.
      • Goossens V.
      • Kokkali G.
      • Meijer-Hoogeveen M.
      • Coonen E.
      • Moutou C.
      ESHRE PGD Consortium data collection XIV-XV: cycles from January 2011 to December 2012 with pregnancy follow-up to October 2013.
      ). The exclusion of embryos due to a mutation can result in no embryos available for transfer. One study reported 39.8% of cycles performed with PGT-M and aneuploidy resulted in no transferable embryos (
      • Minasi M.G.
      • Fiorentino F.
      • Ruberti A.
      • Biricik A.
      • Cursio E.
      • Cotroneo E.
      • Varricchio M.T.
      • Surdo M.
      • Spinella F.
      • Greco E.
      Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a successful clinical approach.
      ). HHGE could improve the efficiency of PGT-M by increasing the number of transferrable embryos, thus decreasing the need for multiple IVF cycles with its associated physical risks and costs. Based on the percentage of cycles with no transferable embryos due to a genetic mutation, we estimate that ∼3,000 IVF cycles annually would benefit from this approach in the US alone (
      • Viotti M.
      • Victor A.R.
      • Griffin D.K.
      • Groob J.S.
      • Brake A.J.
      • Zouves C.G.
      • Barnes F.L.
      Estimating Demand for Germline Genome Editing: An In Vitro Fertilization Clinic Perspective.
      ;
      • Minasi M.G.
      • Fiorentino F.
      • Ruberti A.
      • Biricik A.
      • Cursio E.
      • Cotroneo E.
      • Varricchio M.T.
      • Surdo M.
      • Spinella F.
      • Greco E.
      Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a successful clinical approach.
      ).
      Another common argument against HHGE is that somatic gene therapy or drugs provide a safer path to therapy without risks to the germline (
      • Lanphier E.
      • Urnov F.
      • Haecker S.E.
      • Werner M.
      • Smolenski J.
      Don’t edit the human germ line.
      ). While somatic gene therapy trials are encouraging, limitations to effective therapy include an immune barrier and treatment after irreversible damage from disease onset has occurred. In utero gene therapy may provide a window of opportunity for effective treatment due to small fetal size, tolerogenic fetal immune system, accessible stem and/or progenitor cells, permeable blood-brain barrier, and potential to treat before disease onset, critical for diseases with high prenatal or perinatal morbidity and mortality (
      • Palanki R.
      • Peranteau W.H.
      • Mitchell M.J.
      Delivery technologies for in utero gene therapy.
      ). The clinical utility of in utero gene therapy has so far been limited by poor efficacy and safety concerns, including that a therapy may rescue a pregnancy but still result in a child with disease. Effective somatic or in utero gene therapies may in fact increase the need for germline intervention as couples with homozygous mutations will be more common. For instance, patients with cystic fibrosis now live long enough to raise a family and may wish to avoid passing on the disease to their children. Hence, effective somatic gene therapy does not obviate gene therapy of the germline. A patient who is a carrier for a disease-causing mutation may also wish to avoid passing on the mutation to help prevent the risk of disease in subsequent generations. This could expand the utility of HHGE to millions of couples.
      Embryos with chromosomal aneuploidy are more commonly encountered than embryos with homozygous disease-causing mutations. The number of IVF-generated aneuploid embryos increases exponentially with increasing maternal age, reaching 85% of all embryos by the age of 43 (
      • Franasiak J.M.
      • Forman E.J.
      • Hong K.H.
      • Werner M.D.
      • Upham K.M.
      • Treff N.R.
      • Scott R.T.
      Aneuploidy across individual chromosomes at the embryonic level in trophectoderm biopsies: changes with patient age and chromosome structure.
      ). Intriguing new data demonstrated the possibility of targeting and deleting an entire chromosome in both mouse and human embryos using Cas9 (
      • Adikusuma F.
      • Williams N.
      • Grutzner F.
      • Hughes J.
      • Thomas P.
      Targeted Deletion of an Entire Chromosome Using CRISPR/Cas9.
      ;
      • Zuo E.
      • Huo X.
      • Yao X.
      • Hu X.
      • Sun Y.
      • Yin J.
      • He B.
      • Wang X.
      • Shi L.
      • Ping J.
      • et al.
      CRISPR/Cas9-mediated targeted chromosome elimination.
      ;
      • Zuccaro M.V.
      • Xu J.
      • Mitchell C.
      • Marin D.
      • Zimmerman R.
      • Rana B.
      • Weinstein E.
      • King R.T.
      • Palmerola K.L.
      • Smith M.E.
      • et al.
      Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.
      ). This potential use of Cas9 will still require extensive basic research to investigate the mechanisms of chromosome loss and the risk of adverse outcomes, including retention of chromosome segments that could cause developmental issues.
      • Viotti M.
      • Victor A.R.
      • Griffin D.K.
      • Groob J.S.
      • Brake A.J.
      • Zouves C.G.
      • Barnes F.L.
      Estimating Demand for Germline Genome Editing: An In Vitro Fertilization Clinic Perspective.
      estimated that 20% of cycles produce only aneuploid embryos, which in the United States alone amounts to ∼17,600 cycles. Approximately 5% of human oocytes contain single-chromosome gains (
      • McCoy R.C.
      • Demko Z.P.
      • Ryan A.
      • Banjevic M.
      • Hill M.
      • Sigurjonsson S.
      • Rabinowitz M.
      • Petrov D.A.
      Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development.
      ); these oocytes may be the most amenable to correction of chromosomal content.
      Patients with a Robertsonian chromosome translocation could also benefit from this technology. Robertsonian translocations are the most common form of chromosomal translocations in humans, identified in approximately 1 in 1,000 individuals (
      • Hamerton J.L.
      • Canning N.
      • Ray M.
      • Smith S.
      A cytogenetic survey of 14,069 newborn infants. I. Incidence of chromosome abnormalities.
      ). It occurs when the long q arms of two acrocentric chromosomes merge by translocation, and their short p arms are lost. Because the lost short p arms do not contain unique genetic sequences, these individuals are typically phenotypically normal but are at increased risk for recurrent pregnancy loss due to embryonic aneuploidy. Individuals with a Robertsonian 21q;21q translocation, in particular, may benefit from removing a chromosome since essentially all resulting offspring have trisomy 21, which carries an 85% risk of pregnancy loss (
      • Ercis M.
      • Balci S.
      Can a parent with balanced Robertsonian translocation t(21q;21q) have a non-Down’s offspring?.
      ). Approximately 2% of individuals with trisomy 21 are due to a Robertsonian 21q;21q translocation (
      • Mutton D.
      • Alberman E.
      • Hook E.B.
      National Down Syndrome Cytogenetic Register and the Association of Clinical Cytogeneticists
      Cytogenetic and epidemiological findings in Down syndrome, England and Wales 1989 to 1993.
      ). While years of extensive preclinical testing is still needed to determine feasibility and safety, both gene editing and correction of aneuploidies have the potential to benefit the health of the embryo and the resulting child.

      Non-maleficence: Risks and safety

      Rigorous affirmation of the scientific proof of concept and the copious preclinical evidence to establish the knowledge of risks and benefits is essential prior to clinical translation. Genome editing carries risks beyond those incurred by natural reproduction or IVF alone. Mosaicism and off-target effects are major concerns. Genetic engineering of an embryo or gamete without a mutation would mean to expose it to risk of off-target effects without any benefits to the resulting child. Accordingly, the Royal Society and the National Academies of Sciences, Engineering, and Medicine limit the use of genome editing to only affected embryos with known pathogenic variants (
      NAS, National Academy of Medicine, National Academy of Sciences and the Royal Society
      Heritable Human Genome Editing.
      ). Identifying which embryos to target for genetic modification is a challenge, in particular at the earliest stages when genome editing can be performed on one copy to avoid mosaicism. In studies involving the fertilization of oocytes with sperm from a donor with a heterozygous mutation, all embryos, including wild-type embryos, were injected with Cas9 (
      • Ma H.
      • Marti-Gutierrez N.
      • Park S.W.
      • Wu J.
      • Lee Y.
      • Suzuki K.
      • Koski A.
      • Ji D.
      • Hayama T.
      • Ahmed R.
      • et al.
      Correction of a pathogenic gene mutation in human embryos.
      ;
      • Tang L.
      • Zeng Y.
      • Du H.
      • Gong M.
      • Peng J.
      • Zhang B.
      • Lei M.
      • Zhao F.
      • Wang W.
      • Li X.
      • Liu J.
      CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.
      ) or with a base editor (
      • Zeng Y.
      • Li J.
      • Li G.
      • Huang S.
      • Yu W.
      • Zhang Y.
      • Chen D.
      • Chen J.
      • Liu J.
      • Huang X.
      Correction of the Marfan Syndrome Pathogenic FBN1 Mutation by Base Editing in Human Cells and Heterozygous Embryos.
      ;
      • Liang P.
      • Ding C.
      • Sun H.
      • Xie X.
      • Xu Y.
      • Zhang X.
      • Sun Y.
      • Xiong Y.
      • Ma W.
      • Liu Y.
      • et al.
      Correction of β-thalassemia mutant by base editor in human embryos.
      ). For oocytes, though not for sperm, it is possible to infer the genetic content through analysis of the polar bodies. Thus, HHGE in the embryo may be most applicable to the female germline and to homozygous mutations in the male germline. While stem cell-derived gametes would allow for extensive genetic analysis prior to embryo generation, in vitro-derived gametes also add enormous new risks. In vitro gametogenesis, in particular, from induced pluripotent stem cells combines an extensive sequence of cellular manipulations. The associated risks are numerous from incomplete reprogramming to genetic and epigenetic changes.

      Non-maleficence: Exploitation for non-therapeutic modification

      Some are concerned that HHGE may lead to a “slippery slope” going beyond disease prevention to select for desirable traits. This has been referred to as enhancement or “designer babies.” While some argue that this can be controlled through policy and regulation, others worry that perceived enhancement technologies will be used in other countries without sufficient regulations or oversight (
      Ethics Committee of the American Society for Reproductive Medicine
      Ethics in embryo research: a position statement by the ASRM Ethics in Embryo Research Task Force and the ASRM Ethics Committee.
      ). Beyond the interests of parents and the health of a child, genetic diversity is the principal asset of human reproduction. It is the basis for phenotypic diversity and thereby contributes to the formation of a highly complex society. This diversity could be compromised if pressures experienced by parents are projected to the genotypes of their children. It is also difficult to know which, among the innumerable normal variants, prepares their children best for the future.

      Autonomy: Reproductive rights and lack of ability to consent before birth

      Given alternative paths to parenthood including adoption or gamete donation, the desire for genetic relatedness will be weighed against the risks of HHGE. Risks and benefits of existing and novel reproductive treatments impact both parents and child with an important distinction; the prospective parents can evaluate risks and benefits and consent to them, while the human being to be created cannot. Still others assert parents make countless decisions that shape their children’s future and that the parental desire to enhance the health and happiness of their children is an existing and indeed admirable aspect of parenthood, which often begins prior to conception and continues throughout the child’s life (
      Ethics Committee of the American Society for Reproductive Medicine
      Ethics in embryo research: a position statement by the ASRM Ethics in Embryo Research Task Force and the ASRM Ethics Committee.
      ).

      Justice: Equitable access and allocation of resources

      Additional concerns within the debate on the ethics of HHGE relate to inequality. As with many reproductive technologies, HHGE may only be accessible to individuals and countries who can afford it. This reality may increase health disparities among socioeconomic classes (
      Nuffield Council on Bioethics
      Genome Editing and Human Reproduction: Social and Ethical Issues.
      ). Others argue that HHGE could instead balance the inequalities brought on by genetic diseases and variants (
      • Gyngell C.
      • Bowman-Smart H.
      • Savulescu J.
      Moral reasons to edit the human genome: picking up from the Nuffield report.
      ;
      • De Wert G.
      • Heindryckx B.
      • Pennings G.
      • Clarke A.
      • Eichenlaub-Ritter U.
      • van El C.G.
      • Forzano F.
      • Goddijn M.
      • Howard H.C.
      • Radojkovic D.
      • et al.
      European Society of Human Genetics and the European Society of Human Reproduction and Embryology
      Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE.
      ). Data on human genetic diversity and the role of gene variants under different genomic and external environments are fundamental to gene editing. This knowledge is dependent on genetic data from diverse ethnic backgrounds and environments. Some argue that the current genetic repositories are not representative of the global population (
      • Cavaliere G.
      The Ethics of Human Genome Editing. WHO Expert Advisory Committee on Developing Global Standards for Governance and Oversight of Human Genome Editing, Background Paper.
      ). Costs and allocation of resources is also a consideration. For reproductive treatments, the expenses are generated at the beginning; the costs of managing chronic medical conditions, however, may be significantly greater in comparison due to repeated hospitalization, testing, and treatment.

      Embryo research and destruction

      Preclinical evidence of efficacy and safety of HHGE requires a large body of research. Preclinical research on genome editing in human embryos is incompatible with their use in reproduction, resulting in their destruction. For some, such outcome is ethically unacceptable on the grounds that embryos should be granted the full rights of a living person. For some patients, IVF and the discarding of embryos also pose moral and ethical dilemmas. These concerns can be further augmented after preimplantation testing reveals one or more of the embryos in question contain a disease-causing mutation. Theoretically, HHGE could repair and salvage the diseased embryos that otherwise would be discarded. However, while beneficial for some embryos, HHGE is unlikely to result in the transfer of every embryo created in an IVF cycle. Technologies such as HHGE and PGT-M could also play a role in avoiding pregnancy termination based on the discovery of a genetic anomaly through prenatal testing (
      Ethics Committee of the American Society for Reproductive Medicine.
      Use of preimplantation genetic testing for monogenic defects (PGT-M) for adult-onset conditions: an Ethics Committee opinion.
      ). For some, the discarding of embryos is more acceptable than prenatal diagnosis followed by abortion (
      • Cameron C.
      • Williamson R.
      Is there an ethical difference between preimplantation genetic diagnosis and abortion?.
      ).

      Regulation

      HHGE is largely forbidden globally by laws and regulations. A 2020 policy survey found that the majority of countries (96 of 106) surveyed have policy documents—legislation, regulations, guidelines, codes, and international treaties—relevant to the use of HHGE (
      • Baylis F.
      • Darnovsky M.
      • Hasson K.
      • Krahn T.M.
      Human Germ Line and Heritable Genome Editing: The Global Policy Landscape.
      ). No country explicitly permits the use of genetically modified embryos for reproductive intent. Five countries (Colombia, Panama, Belgium, Italy, and United Arab Emirates) would allow potential exceptions for HHGE and therapeutic purposes. For instance, Colombia allows for HHGE if “aimed at relieving suffering or improving the health of the person and humanity” (
      • Baylis F.
      • Darnovsky M.
      • Hasson K.
      • Krahn T.M.
      Human Germ Line and Heritable Genome Editing: The Global Policy Landscape.
      ).
      National policies surrounding germline editing for research purposes without reproductive intent are much more mixed. Most surveyed countries do not have specific regulation, either permissive or prohibitive. Seventeen countries, including Canada, Germany, Brazil, and Switzerland, do not permit human germline genome research. Alternatively, 12 countries, including the United States, the United Kingdom, Japan, China, Sweden, Ireland, Norway, Thailand, Iran, Congo, Burundi, and India, permit research on human germline genome editing for research without reproductive intent (
      • Baylis F.
      • Darnovsky M.
      • Hasson K.
      • Krahn T.M.
      Human Germ Line and Heritable Genome Editing: The Global Policy Landscape.
      ).
      In the United States, scientific research into the editing of the genome of the human embryo without transfer for reproductive purposes remains permissible although ineligible for public funding. The Food and Drug Administration (FDA) is also prohibited from considering applications for clinical trials “in which a human embryo is intentionally created or modified to include heritable genetic modification” (
      Congress.Gov
      Public Law 114-92. Consolidated Appropriations Act, 2016. Sec 749. December 18, 2015.
      ). Of note, no legislator has stated whether the language of the moratorium applies to editing of oocytes, sperm, or gamete precursors (
      • Cohen I.G.
      • Sherkow J.S.
      • Adashi E.Y.
      Gene Editing Sperm and Eggs (not Embryos): Does it Make a Legal or Ethical Difference?.
      ). These regulations may all have similar intent: to prevent inappropriate or premature use of a technology with transformative potential. While all nations aim to improve human health, complete prohibitions limit the potential for translational research and demonstrate the lack of public confidence in regulation’s ability to distinguish appropriate from inappropriate use, both of which have yet to be defined. Future policy discussions should continue to engage the scientific, medical, and public communities to reflect shared national interests and define acceptable use.

      Conclusions

      Science and imagination possess the freedom to reach beyond what can be translated into clinical practice for the purpose of better understanding human biology, reproduction, and genetics. While science continues to rapidly advance, in order to arrive at decisions with far-reaching implications, the public, medical, and scientific community should be engaged in meaningful discussions regarding the pursuit and potential of these powerful reproductive tools.
      The United Kingdom’s handling of mitochondrial replacement therapy may serve as a model in this context (
      • Claiborne A.B.
      • English R.A.
      • Kahn J.P.
      ETHICS OF NEW TECHNOLOGIES. Finding an ethical path forward for mitochondrial replacement.
      ). Years of preclinical basic research preceded its consideration for use in human reproduction. At the same time, the United Kingdom actively sought the public’s opinion to help inform policymakers, industry, and the research community. This led to state-sanctioned clinical trials and could be used as a model for the ethically defensible and publicly acceptable pursuit of HHGE. In stark contrast to the birth of the two girls with edited CCR5 genes in 2018, decisions to move forward with mitochondrial replacement were not made by individual scientists or doctors, but by independent regulators and the public. This path takes more time but will likely have a longer-lasting benefit to patients. For instance, while a mitochondrial replacement procedure was successfully performed in Mexico in 2017 (
      • Zhang J.
      • Liu H.
      • Luo S.
      • Lu Z.
      • Chávez-Badiola A.
      • Liu Z.
      • Yang M.
      • Merhi Z.
      • Silber S.J.
      • Munné S.
      • et al.
      Live birth derived from oocyte spindle transfer to prevent mitochondrial disease.
      ), apparently based on institutional rather than state-level oversight, no additional cases have since been reported. Alternatively, state-sanctioned mitochondrial donation studies are ongoing in the United Kingdom. While HHGE has the potential to transform the field of reproductive medicine, it is the consensus of many (
      • Plaza Reyes A.
      • Lanner F.
      Towards a CRISPR view of early human development: applications, limitations and ethical concerns of genome editing in human embryos.
      ;
      • Rossant J.
      Gene editing in human development: ethical concerns and practical applications.
      ;
      • Adashi E.Y.
      • Cohen I.G.
      Therapeutic Germline Editing: Sense and Sensibility.
      ;
      • Lea R.A.
      • Niakan K.
      Human germline genome editing.
      ) that great caution must be exercised with an eye on the broader societal and ethical issues that surround its application.

      Acknowledgments

      This work was supported by the NYSTEM award # C32564GG to D.E. J.T. is supported by a clinical fellowship in reproductive endocrinology and infertility.

      Declaration of interests

      D.E. is a member of the Cell Editorial Board. E.Y.A. serves as Co-Chair of the Safety Advisory Board of Ohana Biosciences. J.T. declares no conflicts of interest.

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