Heritable human genome editing: Research progress, ethical considerations, and hurdles to clinical practice Jenna Turocy,1 Eli Y. Adashi,2 and Dieter Egli1,3,4,* 1Department of Obstetrics and Gynecology, Columbia University, New York, NY 10032, USA 2Professor of Medical Science, Brown University, Providence, RI, USA 3Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, NY 10032, USA 4Columbia University Stem Cell Initiative, New York, NY 10032, USA *Correspondence: email@example.com
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 edit- ing, the unique ethical challenges that cannot be overlooked, and the hurdles that must be overcome prior to translating these technologies into clinical practice.
The introduction of genome editing using clustered regularly
interspaced short palindromic repeats (CRISPR)-based tech-
nologies generated tremendous enthusiasm as well as con-
troversy within the medical and public communities. Herita-
ble Human Genome Editing (HHGE) has the potential to
treat or even eradicate genetic diseases. By addressing ge-
netic disease before the defect is amplified through cell pro-
liferation 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 diffi-
cult to manage, let alone cure. This hope provides the ratio-
nale for research, but not yet for therapy, as criteria of effi-
cacy 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 re-
views have also discussed heritable genome editing, empha-
sizing its potential for understanding gene function in the
early human embryo (Lea and Niakan, 2019; Rossant,
2018; Plaza Reyes and Lanner, 2017). 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.
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 and Rothstein, 2013). DNA DSBs may occur during the
DNA replication process, after exposure to ionizing radiation or
chemotherapy, or after experimental manipulation and are re-
paired 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 mu-
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 po-
sition. 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 nucle-
ases (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
Cell 184, March 18, 2021 ª 2021 Elsevier Inc. 1561
Figure 1. Pathways 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.
1562 Cell 184, March 18, 2021
(Gasiunas et al., 2012; Jinek et al., 2012), it was adapted to modi-
fying the genome of cultured mammalian cells (Cong et al., 2013;
Mali et al., 2013) followed by human embryos (Liang et al., 2015;
Kang et al., 2016; Tang et al., 2017) and reviewed in Lea and Nia-
Initial CRISPR-Cas9-mediated studies in human embryos
focused on rates of mutation correction, introduction of off-
target edits, and mosaicism—multiple different genetic out-
comes within the same embryo (Liang et al., 2015; Tang et al.,
2017; Kang et al., 2016) (Figure 2). The first such study made
use of non-viable tripronuclear (3PN) embryos. While this exper-
imental approach took advantage of embryos that would be clin-
ically 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 b gene (HBB) (Liang et al., 2015). 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 et al., 2016). 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 em-
bryos, 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 et al.,
2017). Using diploid 2PN human embryos fertilized with mutant
sperm, Tang et al. (2017) 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
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 et al., 2017). As the sperm donor used was heterozy-
gous, 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 hu-
man 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 et al., 2018). According to Gu et al.
(2018), the efficiency of targeted integration could be further
increased to 95% by tethering a biotinylated DNA repair tem-
plate 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 et al., 2019; Canny et al., 2018). 53BP1 inhibits
DSB repair by HDR and promotes NHEJ (Bunting et al., 2010),
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 ef-
ficiency in mouse embryos (Wilde et al., 2018). However, reliance
on Rad51 could also result in detrimental genetic changes
including translocations due to increased recombination be-
tween homologous sequences in the human genome (Richard-
son et al., 2004). Neither approach has thus far been tested in hu-
Most groups have focused on using HDR to introduce pro-
grammed edits into the human germline genome (Liang et al.,
2015; Kang et al., 2016; Tang et al., 2017; Ma et al.2017). How-
ever, 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 et al., 2020). The gen-
eration of novel alleles may be very useful in a research context,
such as to study the function of a gene product in early embry-
onic development. This experimental approach has been used
to identify a requirement of POU5F1 for normal blastocyst devel-
opment (Fogarty et al., 2017). However, in the context of repro-
duction, 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 com-
mon variants in the relevant population to correct a mutation
(NAS, National Academy of Medicine, National Academy of Sci-
ences and the Royal Society, 2020). Thus, HDR is preferable to
NHEJ as a path to germline gene correction. However, low effi-
ciency 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 et al., 2018). Such deletions are also com-
mon in mouse embryonic stem cells (Kosicki et al., 2018). 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 anal-
ysis in human embryos (Ma et al., 2018). Large deletions were
Cell 184, March 18, 2021 1563
Figure 2. Efficiencies of genome-editing out-
comes 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 tem- plate for EYS. *Number of embryos analyzed with 8 or more cells, from Figure 3a of Fogarty et al., 2017. 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 et al., 2015), CCR5 (Kang et al., 2016), G6PD and HBB in 2PN embryos (Tang et al., 2017), MYBPC3 (Ma et al., 2017), POU5F1 (Alanis-Lobato et al., 2020; Fogarty et al., 2017), and EYS (Zuccaro et al., 2020). 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.
1564 Cell 184, March 18, 2021
similarly missing in another study targeting the EYS locus (Zuc-
caro et al., 2020). Fogarty and colleagues found deletions of
up to �28 bp (Fogarty et al., 2017). Though this observation does not exclude the possibility of large deletions, human em-
bryos do not appear to incur deletions of several hundred base
pairs at the frequency seen in mice, thereby pointing to spe-
cies-specific differences in DSB repair.
Surprisingly, Cas9 resulted in frequent chromosomal changes
in human embryos. Zuccaro and colleagues targeted a blind-
ness-causing mutation in the EYS gene with CRISPR-Cas9
(Zuccaro et al., 2020). 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
et al. (2017) used CRISPR-Cas9 to create mutations in the
POU5F1 gene for embryo developmental studies and also found
chromosomal changes (Alanis-Lobato et al., 2020). The multiple
possible outcomes associated with the generation of a DSB pre-
sent daunting challenges. Chromosomal changes as a conse-
quence of Cas9 cleavage can also occur in human differentiated
cells, though at about 10-fold lower frequency than in human
embryos (Leibowitz et al., 2020), 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
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 et al., 2018)
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 et al., 2020). In somatic
cells, delayed addition of the anti-Crispr protein AcrIIA4 inhibits
off-target cleavage while still allowing on target activity (Shin
et al., 2017). 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 ed-
iting do not use DSBs and thus may avoid some of the aforemen-
tioned undesirable repair outcomes. In base editing, a catalyti-
cally inactive Cas9 serves to guide a deaminase to a specific
site in the genome to mediate site-specific nucleotide to nucleo-
tide conversions (Komor et al., 2016). Base editing has been
tested in human embryos (Zhou et al., 2017; Zhang et al.,
2019; Li et al., 2017;, Zeng et al., 2018) and was found to be high-
ly 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 mech-
anisms to the deaminated base. Studies also reported mosai-
cism 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 et al., 2017) 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 and Liu, 2018). 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 transcrip-
tion into the single-stranded nick (Anzalone et al., 2019). Prime ed-
iting also allows for the repair of a much wider spectrum of
mutations without the need for a donor DNA template or the gen-
eration of a DSB. Using prime editing to correct the genes respon-
sible 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 et al., 2019). 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
et al., 2020; Liu et al., 2020). 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 em-
bryos, and there is insufficient understanding of endogenous DNA
repair pathways in the human embryo to anticipate its associated
MITOCHONDRIAL REPLACEMENT THERAPY
In addition to nuclear DNA mutations, heritable genetic disease
can also be caused by mutations in mitochondrial DNA. Mito-
chondrial 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 preim-
plantation genetic diagnosis or mitochondrial replacement.
Mitochondrial replacement entails the replacement of the
mutated mitochondrial DNA of the oocyte with a healthy mito-
chondrial genome from a donated oocyte (Herbert and Turnbull,
2018). Nuclear content of a maternal oocyte is transferred to a
normal ‘‘enucleated’ oocyte from a donor female prior to implan-
tation. The offspring will have all the nuclear genetic components
of the parents but mitochondrial DNA from a female donor. Mito-
chondrial 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 syn-
drome, resulting in a child with predominantly normal mitochon-
drial DNA (Zhang et al., 2017).
Cell 184, March 18, 2021 1565
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 mitochon-
drial DNA may be an alternative approach to treating mitochon-
drial disease (Reddy et al., 2015). Only recently has it become
possible to edit the mitochondrial genome using TALEN nucle-
ases fused to a deaminase that can act on double-stranded
DNA (Mok et al., 2020). 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 mitochon-
drial 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 intro-
ducing 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 ed-
iting over embryos. They allow for a larger number of modifica-
tions, 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 et al. (2015) reported the editing of mouse spermato-
gonial 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% ef-
ficiency. 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 spermatogen-
esis 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 dis-
ease modeling. For example, Schwank et al. (2013) used
CRISPR-Cas9 to successfully correct cystic fibrosis mutations
in induced pluripotent stem cells derived from cystic fibrosis pa-
tients. 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 et al. (2011) 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
1566 Cell 184, March 18, 2021
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 (Haya-
shi et al., 2012). More recently, Hayashi’s laboratory identified
transcription factors that allow conversion of mouse pluripotent
stem cells to oocyte-like cells that proved fertilization compe-
tent, although they failed to undergo normal meiosis and give
rise to embryos (Hamazaki et al., 2021). Though primordial
germ cell-like cells have been made from human pluripotent
stem cells (Yamashiro et al., 2018), 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 et al., 2016) or
from sperm injected into enucleated oocytes (Zhang et al.,
2020). 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 et al., 2020). Imprinting patterns of
these embryos are indistinguishable from IVF controls, and
gene-expression patterns were very similar, though a small num-
ber of genes of unknown significance were differentially ex-
pressed. 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.
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 et al., 2020), and their genomes have been modified at
scale in the context of genetic screens (Yilmaz et al., 2020).
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 et al., 2012). 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 develop-
ment 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 et al., 2020).
Figure 3. Editing 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 et al., 2016). (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.
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Cell 184, March 18, 2021 1567
Culture-induced genetic or epigenetic change will be clonally in-
herited and remain of unknown functional significance until a hu-
man 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 partic-
ularly true for induced pluripotent stem cells derived from so-
matic cells, which contain mutations of somatic origin as well
as from in vitro reprogramming (Gore et al., 2011; Bhutani
et al., 2016; Rouhani et al., 2016; Yoshihara et al., 2017). 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 con-
cerns. Shortly before the 2018 International Human Genome Ed-
iting Summit, reports emerged that two girls were born after
germline genome editing to prevent the expression of the HIV re-
ceptor CCR5 (Cyranoski and Ledford, 2018). This announce-
ment 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 docu-
ments signed by the prospective parents, and the lack of public
discussion and input regarding the personal and societal conse-
quences of HHGE (Savulescu and Singer, 2019).
Many called for a moratorium on clinical HHGE (Lander et al.,
2019) 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-malefi-
cence, autonomy, and justice (Figure 4).
Beneficence: Therapeutic benefits With over 10,000 known monogenic diseases, heritable diseases
collectively affect roughly 5%–7% of the human population (Ko-
fler and Kraschel, 2018). 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 em-
bryos 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 prev-
Maternal and paternal genomes in the embryo result in a diploid, 46 XX karyotyp attempted. For use of a maternal haploid ESC, its edited genome would replace th and male offspring (not illustrated). Below: tools for genome editing. Cas9 induces or through end joining. Cleavage between regions of microhomology (red nucleo base editing (not illustrated), involve a single strand DNA cut.
1568 Cell 184, March 18, 2021
alence and even eliminate selected heritable diseases. Some
argue that the medical need for HHGE is so compelling that pro-
ceeding with this use of genome editing is a moral imperative
(Gyngell et al., 2019) and that doing so would help to ‘‘lighten
the burden of human existence’’ (Harris, 2016).
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, 2020). Patients
with a genetic disorder can use PGT-M to screen IVF embryos
for the disease-causing gene of interest and transfer only dis-
ease-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 sug-
gests that the clinical need for HHGE for cases that are not
amenable to PGT-M is exceedingly small (Viotti et al., 2019).
An analysis by Viotti et al. (2019) estimated that if all of the pa-
tients 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 effec-
tive for all couples. Even without PGT-M, a limited number of oo-
cytes 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 et al., 2017). The exclusion of embryos due to a muta-
tion can result in no embryos available for transfer. One study re-
ported 39.8% of cycles performed with PGT-M and aneuploidy
resulted in no transferable embryos (Minasi et al., 2017). 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 annu- ally would benefit from this approach in the US alone (Viotti et al.,
2019; Minasi et al., 2017).
Another common argument against HHGE is that somatic gene
germline (Lanphier et al., 2015). While somatic gene therapy trials
are encouraging, limitations to effective therapy include an im-
munebarrierand treatmentafterirreversible damagefromdisease
onset has occurred. In utero gene therapy may provide a window
of opportunity for effective treatment due to small fetal size, tolero-
genic 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 et al., 2021). The clinical utility of
e. The last step, to establish a pregnancy for the birth of a child, has not been e genome of the oocyte, and would be fertilized by sperm, allowing both female a double-stranded break, which can be repaired with a homologous template
tides) can result in predictable outcomes. In contrast, prime editing, as well as
Figure 4. Ethical 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 sur- rounding human heritable genome editing. (A) Benef- icence: 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 out- comes 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) Jus- tice: Access to assisted reproductive technologies is limited to individuals and countries who can afford it (figured adapted from (DeWeerdt, 2020).
Cell 184, March 18, 2021 1569
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 therapiesmayinfactincrease the needforgermlineinterven-
For instance, patients with cystic fibrosis now live long enough to
raise a family and may wish toavoid passing on the disease totheir
children. Hence, effective somatic gene therapy does not obviate
gene therapy of the germline. A patient who is a carrier for a dis-
ease-causing mutation may also wish to avoid passing on the mu-
tation to help prevent the risk of disease in subsequent genera-
tions. 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 in-
creases exponentially with increasing maternal age, reaching
85% of all embryos by the age of 43 (Franasiak et al., 2014).
Intriguing new data demonstrated the possibility of targeting and
deleting an entire chromosome in both mouse and human em-
bryos using Cas9 (Adikusuma et al., 2017; Zuo et al., 2017; Zuc-
caro et al., 2020). This potential use of Cas9 will still require exten-
sive basic research to investigate the mechanisms of
chromosome loss and the risk of adverse outcomes, including
retention of chromosome segments that could cause develop-
mental issues. Viotti et al. (2019) estimated that 20% of cycles pro-
duce only aneuploid embryos, which in the United States alone
amounts to �17,600 cycles. Approximately 5% of human oocytes contain single-chromosome gains (McCoy et al., 2015); these oo-
cytes may be the most amenable to correction of chromosomal
Patients with a Robertsonian chromosome translocation could
also benefit from this technology. Robertsonian translocations
are the most common form of chromosomal translocations in hu-
mans, identified in approximately 1 in 1,000 individuals (Hamer-
ton et al., 1975). It occurs when the long q arms of two acrocen-
tric chromosomes merge by translocation, and their short p arms
are lost. Because the lost short p arms do not contain unique ge-
netic 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 tri-
somy 21, which carries an 85% risk of pregnancy loss (Ercis
and Balci, 1999). Approximately 2% of individuals with trisomy
21 are due to a Robertsonian 21q;21q translocation (Mutton
et al., 1996). 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 ed-
iting carries risks beyond those incurred by natural reproduction
or IVF alone. Mosaicism and off-target effects are major con-
cerns. Genetic engineering of an embryo or gamete without a
mutation would mean to expose it to risk of off-target effects
1570 Cell 184, March 18, 2021
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 Acad-
emy of Medicine, National Academy of Sciences and the Royal
Society, 2020). 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 et al.,
2017; Tang et al., 2017) or with a base editor (Zeng et al., 2018;
Liang et al., 2017). 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 gameto-
genesis, in particular, from induced pluripotent stem cells com-
bines 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 technolo-
gies will be used in other countries without sufficient regulations
or oversight (Ethics Committee of the American Society for
Reproductive Medicine, 2020). 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 so-
ciety. This diversity could be compromised if pressures experi-
enced by parents are projected to the genotypes of their chil-
dren. 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 exist-
ing 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 hu-
man 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 con-
tinues throughout the child’s life (Ethics Committee of the Amer-
ican Society for Reproductive Medicine, 2020).
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,
2018). Others argue that HHGE could instead balance the in-
equalities brought on by genetic diseases and variants (Gyngell
et al., 2019; De Wert et al., 2018). Data on human genetic diver-
sity 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 popula-
tion (Cavaliere, 2019). Costs and allocation of resources is also a
consideration. For reproductive treatments, the expenses are
generated at the beginning; the costs of managing chronic med-
ical conditions, however, may be significantly greater in compar-
ison 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 dis-
carded. 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 dis-
covery of a genetic anomaly through prenatal testing (Ethics
Committee of the American Society for Reproductive Medicine,
2018). For some, the discarding of embryos is more acceptable
than prenatal diagnosis followed by abortion (Cameron and Wil-
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 et al., 2020). 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 per-
son and humanity’’ (Baylis et al., 2020).
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 Can-
ada, 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 et al., 2020).
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 fund-
ing. 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, 2015). Of note, no legis-
lator has stated whether the language of the moratorium applies
to editing of oocytes, sperm, or gamete precursors (Cohen et al.,
2020). These regulations may all have similar intent: to prevent
inappropriate or premature use of a technology with transforma-
tive 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 regu-
lation’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
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 ge-
netics. 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 mean-
ingful 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 et al.,
2016). Years of preclinical basic research preceded its consider-
ation for use in human reproduction. At the same time, the United
Kingdom actively sought the public’s opinion to help inform pol-
icymakers, 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 mitochon-
drial replacement were not made by individual scientists or doc-
tors, but by independent regulators and the public. This path
takes more time but will likely have a longer-lasting benefit to pa-
tients. For instance, while a mitochondrial replacement proced-
ure was successfully performed in Mexico in 2017 (Zhang et al.,
2017), apparently based on institutional rather than state-level
oversight, no additional cases have since been reported. Alter-
natively, 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 and Lanner, 2017; Rossant,
Cell 184, March 18, 2021 1571
2018; Adashi and Cohen, 2020; Lea and Niakan, 2019) that great
caution must be exercised with an eye on the broader societal
and ethical issues that surround its application.
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
Adashi, E.Y., and Cohen, I.G. (2020). Therapeutic Germline Editing: Sense and
Sensibility. Trends Genet. 36, 315–317.
Adikusuma, F., Williams, N., Grutzner, F., Hughes, J., and Thomas, P. (2017).
Targeted Deletion of an Entire Chromosome Using CRISPR/Cas9. Mol. Ther.
Adikusuma, F., Piltz, S., Corbett, M.A., Turvey, M., McColl, S.R., Helbig, K.J.,
Beard, M.R., Hughes, J., Pomerantz, R.T., and Thomas, P.Q. (2018). Large de-
letions induced by Cas9 cleavage. Nature 560, E8–E9.
Aida, T., Wilde, J.J., Yang, L., Hou, Y., Li, M., Xu, D., Lin, J., Qi, P., Lu, Z., and
Feng, G. (2020). Prime editing primarily induces undesired outcomes in mice.
Alanis-Lobato, G., Zohren, J., Mccarthy, A., Fogarty, N.M.E., Kubikova, N.,
Hardman, E., Greco, M., Wells, D., Turner, J.M.A., and Niakan, K.K. (2020).
Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human em-
bryos. bioRxiv, 2020.06.05.135913.
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., and Liu, D.R.
(2019). Search-and-replace genome editing without double-strand breaks or
donor DNA. Nature 576, 149–157.
Baylis, F., Darnovsky, M., Hasson, K., and Krahn, T.M. (2020). Human Germ
Line and Heritable Genome Editing: The Global Policy Landscape. CRISPR
J. 3, 365–377.
Bhutani, K., Nazor, K.L., Williams, R., Tran, H., Dai, H., D�zakula, �Z., Cho, E.H.,
Pang, A.W.C., Rao, M., Cao, H., et al. (2016). Whole-genome mutational
burden analysis of three pluripotency induction methods. Nat. Commun.
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. (2010). 53BP1 inhibits
homologous recombination in Brca1-deficient cells by blocking resection of
DNA breaks. Cell 141, 243–254.
Cameron, C., and Williamson, R. (2003). Is there an ethical difference between
preimplantation genetic diagnosis and abortion? J. Med. Ethics 29, 90–92.
Canny, M.D., Moatti, N., Wan, L.C.K., Fradet-Turcotte, A., Krasner, D., Ma-
teos-Gomez, P.A., Zimmermann, M., Orthwein, A., Juang, Y.C., Zhang, W.,
et al. (2018). Inhibition of 53BP1 favors homology-dependent DNA repair
and increases CRISPR-Cas9 genome-editing efficiency. Nat. Biotechnol. 36,
Cavaliere, G. (2019). The Ethics of Human Genome Editing. WHO Expert Advi-
sory Committee on Developing Global Standards for Governance and Oversight
of Human Genome Editing, Background Paper. https://www.who.int/ethics/
pdf. (Accessed 25 January 2021).
Claiborne, A.B., English, R.A., and Kahn, J.P. (2016). ETHICS OF NEW TECH-
NOLOGIES. Finding an ethical path forward for mitochondrial replacement.
Science 351, 668–670.
1572 Cell 184, March 18, 2021
Cohen, I.G., Sherkow, J.S., and Adashi, E.Y. (2020). Gene Editing Sperm and
Eggs (not Embryos): Does it Make a Legal or Ethical Difference? J. Law Med.
Ethics 48, 619–621.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X.,
Jiang, W., Marraffini, L.A., and Zhang, F. (2013). Multiplex genome engineering
using CRISPR/Cas systems. Science 339, 819–823.
Congress.Gov (2015). Public Law 114-92. Consolidated Appropriations Act,
2016. Sec 749. December 18, 2015. https://www.congress.gov/bill/
114th-congress/house-bull/2029/text. (Accessed 25 January 2021).
Cyranoski, D., and Ledford, H. (2018). Genome-edited baby claim provokes in-
ternational outcry. Nature 563, 607–608.
De Rycke, M., Goossens, V., Kokkali, G., Meijer-Hoogeveen, M., Coonen, E.,
and Moutou, C. (2017). ESHRE PGD Consortium data collection XIV-XV: cy-
cles from January 2011 to December 2012 with pregnancy follow-up to
October 2013. Hum. Reprod. 32, 1974–1994.
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.; Eu-
ropean Society of Human Genetics and the European Society of Human
Reproduction and Embryology (2018). Responsible innovation in human germ-
line gene editing: Background document to the recommendations of ESHG
and ESHRE. Eur. J. Hum. Genet. 26, 450–470.
DeWeerdt, S. (2020). How much should having a baby cost? Nature 588,
Ercis, M., and Balci, S. (1999). Can a parent with balanced Robertsonian trans-
location t(21q;21q) have a non-Down’s offspring? Lancet 353, 751.
Ethics Committee of the American Society for Reproductive Medicine. (2018).
Use of preimplantation genetic testing for monogenic defects (PGT-M) for
adult-onset conditions: an Ethics Committee opinion. Fertil. Steril. 109,
Ethics Committee of the American Society for Reproductive Medicine (2020).
Ethics in embryo research: a position statement by the ASRM Ethics in Embryo
Research Task Force and the ASRM Ethics Committee. Fertil. Steril. 113,
Fogarty, N.M.E., McCarthy, A., Snijders, K.E., Powell, B.E., Kubikova, N., Bla-
keley, P., Lea, R., Elder, K., Wamaitha, S.E., Kim, D., et al. (2017). Genome ed-
iting reveals a role for OCT4 in human embryogenesis. Nature 550, 67–73.
Franasiak, J.M., Forman, E.J., Hong, K.H., Werner, M.D., Upham, K.M., Treff,
N.R., and Scott, R.T. (2014). Aneuploidy across individual chromosomes at the
embryonic level in trophectoderm biopsies: changes with patient age and
chromosome structure. J. Assist. Reprod. Genet. 31, 1501–1509.
Gasiunas, G., Barrangou, R., Horvath, P., and Siksnys, V. (2012). Cas9-crRNA
ribonucleoprotein complex mediates specific DNA cleavage for adaptive im-
munity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586.
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. (2011). Somatic coding
mutations in human induced pluripotent stem cells. Nature 471, 63–67.
Gu, B., Posfai, E., and Rossant, J. (2018). Efficient generation of targeted large
insertions by microinjection into two-cell-stage mouse embryos. Nat. Bio-
technol. 36, 632–637.
Gyngell, C., Bowman-Smart, H., and Savulescu, J. (2019). Moral reasons to
edit the human genome: picking up from the Nuffield report. J. Med. Ethics
Hamazaki, N., Kyogoku, H., Araki, H., Miura, F., Horikawa, C., Hamada, N.,
Shimamoto, S., Hikabe, O., Nakashima, K., Kitajima, T.S., et al. (2021). Recon-
stitution of the oocyte transcriptional network with transcription factors. Nature
Hamerton, J.L., Canning, N., Ray, M., and Smith, S. (1975). A cytogenetic sur-
vey of 14,069 newborn infants. I. Incidence of chromosome abnormalities.
Clin. Genet. 8, 223–243.
Harris, J. (2016). Germline Modification and the Burden of Human Existence.
Camb. Q. Healthc. Ethics 25, 6–18.
Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., and Saitou, M. (2011). Recon-
stitution of the mouse germ cell specification pathway in culture by pluripotent
stem cells. Cell 146, 519–532.
Hayashi, K., Ogushi, S., Kurimoto, K., Shimamoto, S., Ohta, H., and Saitou, M.
(2012). Offspring from oocytes derived from in vitro primordial germ cell-like
cells in mice. Science 338, 971–975.
Herbert, M., and Turnbull, D. (2018). Progress in mitochondrial replacement
therapies. Nat. Rev. Mol. Cell Biol. 19, 71–72.
Jasin, M., and Rothstein, R. (2013). Repair of strand breaks by homologous
recombination. Cold Spring Harb. Perspect. Biol. 5, a012740.
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentier,
E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive
bacterial immunity. Science 337, 816–821.
Kang, X., He, W., Huang, Y., Yu, Q., Chen, Y., Gao, X., Sun, X., and Fan, Y.
(2016). Introducing precise genetic modifications into human 3PN embryos
by CRISPR/Cas-mediated genome editing. J. Assist. Reprod. Genet. 33,
Kim, Y.B., Komor, A.C., Levy, J.M., Packer, M.S., Zhao, K.T., and Liu, D.R.
(2017). Increasing the genome-targeting scope and precision of base editing
with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35,
Kofler, N., and Kraschel, K.L. (2018). Treatment of heritable diseases using
CRISPR: Hopes, fears, and reality. Semin. Perinatol. 42, 515–521.
Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., and Liu, D.R. (2016). Pro-
grammable editing of a target base in genomic DNA without double-stranded
DNA cleavage. Nature 533, 420–424.
Kosicki, M., Tomberg, K., and Bradley, A. (2018). Repair of double-strand
breaks induced by CRISPR-Cas9 leads to large deletions and complex rear-
rangements. Nat. Biotechnol. 36, 765–771.
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., and Cuppen, E.
(2020). The mutational impact of culturing human pluripotent and adult stem
cells. Nat. Commun. 11, 2493.
Lander, E.S., Baylis, F., Zhang, F., Charpentier, E., Berg, P., Bourgain, C., Frie-
drich, B., Joung, J.K., Li, J., Liu, D., et al. (2019). Adopt a moratorium on her-
itable genome editing. Nature 567, 165–168.
Lanphier, E., Urnov, F., Haecker, S.E., Werner, M., and Smolenski, J. (2015).
Don’t edit the human germ line. Nature 519, 410–411.
Lea, R.A., and Niakan, K. (2019). Human germline genome editing. Nat. Cell
Biol. 21, 1479–1489.
Leibowitz, M.L., Papathanasiou, S., Doerfler, P.A., Blaine, L.J., Yao, Y., Zhang,
C.-Z., Weiss, M.J., and Pellman, D. (2020). Chromothripsis as an on-target
consequence of CRISPR-Cas9 genome editing. bioRxiv, 2020.07.13.200998.
Li, G., Liu, Y., Zeng, Y., Li, J., Wang, L., Yang, G., Chen, D., Shang, X., Chen, J.,
Huang, X., and Liu, J. (2017). Highly efficient and precise base editing in dis-
carded human tripronuclear embryos. Protein Cell 8, 776–779.
Liang, P., Xu, Y., Zhang, X., Ding, C., Huang, R., Zhang, Z., Lv, J., Xie, X., Chen,
Y., Li, Y., et al. (2015). CRISPR/Cas9-mediated gene editing in human tripronu-
clear zygotes. Protein Cell 6, 363–372.
Liang, P., Ding, C., Sun, H., Xie, X., Xu, Y., Zhang, X., Sun, Y., Xiong, Y., Ma, W.,
Liu, Y., et al. (2017). Correction of b-thalassemia mutant by base editor in hu-
man embryos. Protein Cell 8, 811–822.
Liu, Y., Li, X., He, S., Huang, S., Li, C., Chen, Y., Liu, Z., Huang, X., and Wang,
X. (2020). Efficient generation of mouse models with the prime editing system.
Cell Discov. 6, 27.
Ma, H., Marti-Gutierrez, N., Park, S.W., Wu, J., Lee, Y., Suzuki, K., Koski, A., Ji,
D., Hayama, T., Ahmed, R., et al. (2017). Correction of a pathogenic gene mu-
tation in human embryos. Nature 548, 413–419.
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. (2018). Ma et al. reply. Nature
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 (2012). A systematic survey of loss-of-
function variants in human protein-coding genes. Science 335, 823–828.
Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E.,
and Church, G.M. (2013). RNA-guided human genome engineering via Cas9.
Science 339, 823–826.
McCoy, R.C., Demko, Z.P., Ryan, A., Banjevic, M., Hill, M., Sigurjonsson, S.,
Rabinowitz, M., and Petrov, D.A. (2015). Evidence of Selection against Com-
plex Mitotic-Origin Aneuploidy during Preimplantation Development. PLoS
Genet. 11, e1005601.
Minasi, M.G., Fiorentino, F., Ruberti, A., Biricik, A., Cursio, E., Cotroneo, E.,
Varricchio, M.T., Surdo, M., Spinella, F., and Greco, E. (2017). Genetic dis-
eases and aneuploidies can be detected with a single blastocyst biopsy: a suc-
cessful clinical approach. Hum. Reprod. 32, 1770–1777.
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. (2020). A bacterial
cytidine deaminase toxin enables CRISPR-free mitochondrial base editing.
Nature 583, 631–637.
Mutton, D., Alberman, E., and Hook, E.B.; National Down Syndrome Cytoge-
netic Register and the Association of Clinical Cytogeneticists (1996). Cytoge-
netic and epidemiological findings in Down syndrome, England and Wales
1989 to 1993. J. Med. Genet. 33, 387–394.
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. (2019). Stim-
ulation of CRISPR-mediated homology-directed repair by an engineered
RAD18 variant. Nat. Commun. 10, 3395.
NAS, National Academy of Medicine, National Academy of Sciences and the
Royal Society (2020). Heritable Human Genome Editing (The National Acade-
Nuffield Council on Bioethics (2018). Genome Editing and Human Reproduc-
tion: Social and Ethical Issues (Nuffield Council on Bioethics).
Palanki, R., Peranteau, W.H., and Mitchell, M.J. (2021). Delivery technologies
for in utero gene therapy. Adv. Drug Deliv. Rev. 169, 51–62.
Pawluk, A., Davidson, A.R., and Maxwell, K.L. (2018). Anti-CRISPR: discovery,
mechanism and function. Nat. Rev. Microbiol. 16, 12–17.
Plaza Reyes, A., and Lanner, F. (2017). Towards a CRISPR view of early human
development: applications, limitations and ethical concerns of genome editing
in human embryos. Development 144, 3–7.
Reddy, P., Ocampo, A., Suzuki, K., Luo, J., Bacman, S.R., Williams, S.L., Su-
gawara, A., Okamura, D., Tsunekawa, Y., Wu, J., et al. (2015). Selective elim-
ination of mitochondrial mutations in the germline by genome editing. Cell 161,
Rees, H.A., and Liu, D.R. (2018). Base editing: precision chemistry on the
genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788.
Richardson, C., Stark, J.M., Ommundsen, M., and Jasin, M. (2004). Rad51
overexpression promotes alternative double-strand break repair pathways
and genome instability. Oncogene 23, 546–553.
Rossant, J. (2018). Gene editing in human development: ethical concerns and
practical applications. Development 145, dev150888.
Rouhani, F.J., Nik-Zainal, S., Wuster, A., Li, Y., Conte, N., Koike-Yusa, H., Ku-
masaka, N., Vallier, L., Yusa, K., and Bradley, A. (2016). Mutational History of a
Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells. PLoS
Genet. 12, e1005932.
Safier, L.Z., Zuccaro, M.V., and Egli, D. (2020). Efficient SNP editing in haploid
human pluripotent stem cells. J. Assist. Reprod. Genet. 37, 735–745.
Sagi, I., Chia, G., Golan-Lev, T., Peretz, M., Weissbein, U., Sui, L., Sauer, M.V.,
Yanuka, O., Egli, D., and Benvenisty, N. (2016). Derivation and differentiation of
haploid human embryonic stem cells. Nature 532, 107–111.
Savulescu, J., and Singer, P. (2019). An ethical pathway for gene editing.
Bioethics 33, 221–222.
Cell 184, March 18, 2021 1573
Schwank, G., Koo, B.K., Sasselli, V., Dekkers, J.F., Heo, I., Demircan, T., Sa-
saki, N., Boymans, S., Cuppen, E., van der Ent, C.K., et al. (2013). Functional
repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic
fibrosis patients. Cell Stem Cell 13, 653–658.
Shin, J., Jiang, F., Liu, J.-J., Bray, N.L., Rauch, B.J., Baik, S.H., Nogales, E.,
Bondy-Denomy, J., Corn, J.E., and Doudna, J.A. (2017). Disabling Cas9 by
an anti-CRISPR DNA mimic. Sci. Adv. 3, e1701620.
Tang, L., Zeng, Y., Du, H., Gong, M., Peng, J., Zhang, B., Lei, M., Zhao, F.,
Wang, W., Li, X., and Liu, J. (2017). CRISPR/Cas9-mediated gene editing in hu-
man zygotes using Cas9 protein. Mol. Genet. Genomics 292, 525–533.
Viotti, M., Victor, A.R., Griffin, D.K., Groob, J.S., Brake, A.J., Zouves, C.G., and
Barnes, F.L. (2019). Estimating Demand for Germline Genome Editing: An
In Vitro Fertilization Clinic Perspective. CRISPR J. 2, 304–315.
Wilde, J.J., Aida, T., Wienisch, M., Zhang, Q., Qi, P., and Feng, G. (2018). Effi-
cient Zygotic Genome Editing via RAD51-Enhanced Interhomolog Repair. bio-
Wu, Y., Zhou, H., Fan, X., Zhang, Y., Zhang, M., Wang, Y., Xie, Z., Bai, M., Yin,
Q., Liang, D., et al. (2015). Correction of a genetic disease by CRISPR-Cas9-
mediated gene editing in mouse spermatogonial stem cells. Cell Res. 25,
Yamashiro, C., Sasaki, K., Yabuta, Y., Kojima, Y., Nakamura, T., Okamoto, I.,
Yokobayashi, S., Murase, Y., Ishikura, Y., Shirane, K., et al. (2018). Generation
of human oogonia from induced pluripotent stem cells in vitro. Science 362,
Yilmaz, A., Braverman-Gross, C., Bialer-Tsypin, A., Peretz, M., and Benve-
nisty, N. (2020). Mapping Gene Circuits Essential for Germ Layer Differentia-
tion via Loss-of-Function Screens in Haploid Human Embryonic Stem Cells.
Cell Stem Cell 27, 679–691.
1574 Cell 184, March 18, 2021
Yoshihara, M., Araki, R., Kasama, Y., Sunayama, M., Abe, M., Nishida, K., Ka-
waji, H., Hayashizaki, Y., and Murakawa, Y. (2017). Hotspots of De Novo Point
Mutations in Induced Pluripotent Stem Cells. Cell Rep. 21, 308–315.
Zeng, Y., Li, J., Li, G., Huang, S., Yu, W., Zhang, Y., Chen, D., Chen, J., Liu, J.,
and Huang, X. (2018). Correction of the Marfan Syndrome Pathogenic FBN1
Mutation by Base Editing in Human Cells and Heterozygous Embryos. Mol.
Ther. 26, 2631–2637.
Zhang, J., Liu, H., Luo, S., Lu, Z., Chávez-Badiola, A., Liu, Z., Yang, M., Merhi,
Z., Silber, S.J., Munné, S., et al. (2017). Live birth derived from oocyte spindle
transfer to prevent mitochondrial disease. Reprod. Biomed. Online 34,
Zhang, M., Zhou, C., Wei, Y., Xu, C., Pan, H., Ying, W., Sun, Y., Sun, Y., Xiao,
Q., Yao, N., et al. (2019). Human cleaving embryos enable robust homozygotic
nucleotide substitutions by base. Genome Biol. 20, 101.
Zhang, X.M., Wu, K., Zheng, Y., Zhao, H., Gao, J., Hou, Z., Zhang, M., Liao, J.,
Zhang, J., Gao, Y., et al. (2020). In vitro expansion of human sperm through nu-
clear transfer. Cell Res. 30, 356–359.
Zhou, C., Zhang, M., Wei, Y., Sun, Y., Sun, Y., Pan, H., Yao, N., Zhong, W., Li,
Y., Li, W., et al. (2017). Highly efficient base editing in human tripronuclear zy-
gotes. Protein Cell 8, 772–775.
Zuccaro, M.V., Xu, J., Mitchell, C., Marin, D., Zimmerman, R., Rana, B., Wein-
stein, E., King, R.T., Palmerola, K.L., Smith, M.E., et al. (2020). Allele-Specific
Chromosome Removal after Cas9 Cleavage in Human Embryos. Cell 183,
Zuo, E., Huo, X., Yao, X., Hu, X., Sun, Y., Yin, J., He, B., Wang, X., Shi, L., Ping,
J., et al. (2017). CRISPR/Cas9-mediated targeted chromosome elimination.
Genome Biol. 18, 224.
- Heritable human genome editing: Research progress, ethical considerations, and hurdles to clinical practice
- DNA double-strand breaks allow genome editing in human embryos
- Adverse consequences of DNA breaks in human embryos
- Mitochondrial replacement therapy
- Editing of in vitro-derived gametes and embryos
- Spermatogonial stem cells
- Induced pluripotent stem cells
- Haploid human stem cells
- Ethical considerations of heritable human genome editing
- Beneficence: Therapeutic benefits
- Non-maleficence: Risks and safety
- Non-maleficence: Exploitation for non-therapeutic modification
- Autonomy: Reproductive rights and lack of ability to consent before birth
- Justice: Equitable access and allocation of resources
- Embryo research and destruction
- Declaration of interests