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Heritable human genome editing:

Heritable human genome editing:

Leading Edge


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: de2220@cumc.columbia.edu




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.


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




ll Review

(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-

kan (2019).

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-

man embryos.

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).


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




ll Review

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.


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



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).

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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.


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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ll Review

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.


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




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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).



ll Review

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-

liamson, 2003).


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

acceptable use.


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



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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.


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



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  • 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
    • Conclusions
    • Acknowledgments
    • Declaration of interests
    • References

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