Genetics: Current Commentary
Genetically Modified Babies and a First
Application of Clustered Regularly
Interspaced Short Palindromic
Repeats (CRISPR-Cas9)
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Bruce I. Rose, MD, PhD, and Samuel Brown, MD
The world’s first babies with CRISPR-Cas9 (Clustered
Regularly Interspaced Short Palindromic Repeats)–
edited genes were born on November 25, 2018. Dr.
Jiankui He of Southern University of Science and Tech-
nology in Shenzhen performed this gene editing. Dr. He’s
objectives and an assessment of how well they were
achieved are discussed in the context of existing research
in this area.
(Obstet Gynecol 2019;134:157–62)
DOI: 10.1097/AOG.0000000000003327
O n November 26, 2018, at a gene editing con-
ference in Hong Kong, Dr. Jiankui He
announced the delivery of twins Lulu and Nina, con-
ceived with embryos that had been genetically mod-
ified using Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR-Cas9). Dr. He re-
ported that he altered the gene CCR5 in those
embryos. This gene produces a protein that most
strains of human immunodeficiency virus (HIV)
From the Brown Fertility and the Department of Obstetrics and Gynecology,
University of Florida College of Medicine, Jacksonville, Florida
The authors thank Kate Harfe, PhD, for her review of an earlier version of this
manuscript and for her suggestions on how to improve this paper.
Each author has confirmed compliance with the journal’s requirements for
authorship.
Corresponding author: Bruce I. Rose, MD, PhD, Brown Fertility, LLC,
Jacksonville, FL; email: brose@brownfertility.com.
Financial Disclosure
The authors did not report any potential conflicts of interest.
© 2019 The Author(s). Published by Wolters Kluwer Health, Inc. This is an
open-access article distributed under the terms of the Creative Commons
Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND),
where it is permissible to download and share the work provided it is properly
cited. The work cannot be changed in any way or used commercially without
permission from the journal.
ISSN: 0029-7844/19
VOL. 134, NO. 1, JULY 2019
require to enter cells.1 This genetic modification
was done on the embryos to protect the resulting
children from contracting HIV. The children’s father
had HIV.
Although this experiment has not yet been
presented in a peer-reviewed scientific journal, it
has been extensively discussed in both lay and
science-oriented media. It seems that genetic engi-
neering of human embryos is “here” despite its lim-
ited public support. Ed Yong reported in The
Atlantic2 that modifying germ cells before human
implantation is illegal in 15 of 22 countries in West-
ern Europe. In the United States, the U.S. Food and
Drug Administration is prohibited from even review-
ing any drug or biological product involving the
modification of a human embryo.3 The scientific
community has recently expressed hesitation about
the long-term safety of modifying germ cells that will
become babies and has recommended proceeding
cautiously.4–6 The discovery of CRISPR-Cas9 has
made genetic modifications easier to engineer and
less expensive to perform than previously used meth-
ods such as zinc finger nucleases and transcription
activator-like effector nucleases. These older meth-
ods had more complex requirements for program-
ming them to bind to DNA at the targeted site.4,5
Further development of this molecular tool is likely
to make genetic modifications of cells common for
preventing and treating human diseases,3,4 for
manufacturing commercial products that use live or-
ganisms,4,5,7 for exploring the intricacies of the
human genome,8 and even for novel applications
such as the rescue of extinct species.9 We will explain
what CRISPR-Cas9 is and how it was used by Dr. He
to prenatally change the genome of a baby. We will
then look more deeply into the details of this
experiment.
OBSTETRICS & GYNECOLOGY 157
BACKGROUND
The human genome is contained in 23 chromosome
pairs, primarily consisting of DNA. Each DNA
molecule is a dimer containing about six billion
nucleotides connected by a phosphate backbone.
Only four different nucleosides occur in this very
long sequence: adenine (A), guanine (G), thymine (T)
and, cytosine (C). Each of these four nucleotides can
form hydrogen bonds with only one other nucleotide
(A with T and G with C). This is the basis for the
genetic continuity of cells during mitosis or meiosis.
Each strand of the DNA molecule can exactly dictate
the opposite subunit by acting as a template in which
each nucleotide in the sequence establishes hydrogen
bonds with its unique mate and also forms a covalent
bond to the proceeding nucleotide to create an
elongated phosphate spine reconstituting the double-
stranded molecule.
Most life contains DNA, including bacteria and
many viruses. Viruses that attack bacteria, or bacter-
iophages, are very numerous.10 Bacteria have devel-
oped defensive weapons that attach to and destroy
invading bacteriophages by cutting apart their DNA.
In their study of bacteria, scientists have identified
some of these weapons (referred to as restriction en-
zymes) and have used them routinely as tools in the
study of DNA. From different species of bacteria,
more than 3,000 of these bacterial weapons have been
identified. Different restriction enzymes bind to differ-
ent short sequences of DNA (usually 4–8 nucleotides
long) and then make double-stranded cuts in the DNA
near those oligonucleotides.
A more recent discovery in bacteria is a defensive
tool called CRISPR-Cas9 (CRISPR stands for Clus-
tered Regularly Interspaced Short Palindromic Re-
peats.) As with restriction enzymes, CRISPR-Cas9
variants can be found in many bacteria.11 CRISPR-
Cas9 acts like an immune system for its bacterium.12
If a bacterial cell survives an attack by a bacteriophage,
CRISPR-Cas9 retains a small piece of that virus’s
DNA. This retained memory of prior exposure ena-
bles that bacterium to rapidly defend itself, if it is
attacked again by the same species of bacteriophage.
The CRISPR-Cas9 system (Fig. 1), adapted for
genetic engineering differs from restriction enzymes
in that its binding–recognition site is defined by
a longer sequence of about 20 nucleotides. Because
restriction enzyme binding sequences are short, they
bind at many sites and make many cuts in a long
DNA molecule. The binding site of CRISPR-Cas9
molecules is long enough to be used for precise bind-
ing if the CRISPR-Cas9 tool is well designed. Zinc
158 Rose and Brown Genetically Modified Babies Using CRISPR-Cas9
finger nucleases and transcription activator-like effec-
tor nucleases have more complex attachments, which
make them harder to engineer. For example, it takes
many months to create zinc finger nuclease and tran-
scription activator-like effector nuclease editing tools,
whereas it takes days to create a CRISPR-Cas9 tool
(with a cost reduction of similar magnitude).4
Once CRISPR-Cas9 has attached to the DNA
molecule, the CRISPR-Cas9–associated nuclease
Cas9 makes a double-stranded cut in a defined loca-
tion on the DNA molecule. To make this precise cut,
CRISPR-Cas9 must also simultaneously bind to
a short DNA sequence (called a PAM sequence).
The cut is made two or three nucleotides away from
the PAM sequence.5,13 PAM sequences differ and
depend on the bacteria from which the CRISPR-
Cas9 tool was isolated.
CRISPR-Cas9 is guided to its binding site by an
engineered piece of RNA created to attach to the
target location on the DNA on the sense strand.14 The
double-stranded break created by CRISPR-Cas9
needs to be repaired rapidly by the cell or the cell is
likely to die. Prevalent exonucleases will delete nu-
cleotides from bare cut ends of DNA. Random nu-
cleotides may attach to the bare ends. These
deletion and insertion mutations may make the gene’s
protein product nonfunctional. The investigator may
also supply small fragments of DNA that enable the
cell to rebuild a targeted cutting site by using these
pieces as a template in its repair.
CRISPR-Cas9 may be used to cut out a long
sequence of DNA or to remove an entire gene from
a cell’s DNA by using two CRISPR-Cas9 molecules
Fig. 1. Diagram of the CRISPR-Cas9 molecule. Both trans-
activating CRISPR RNA (tracrRNA) and CRISPR (crRNA)
occur in nature. The CRISPR component tracrRNA is
a binding scaffold to the Cas nuclease. The crRNA molecule
is a small molecule that attaches to the RNA derived from
a phage that attacked the bacteria (one of the “spacers” in
CRISPR), which together constitute the guide RNA. Licensed
from Mari-Leaf/Shutterstock.com(SSTK-066F1-A564). Used
with permission. PAM, protospacer adjacent motif.
Rose and Brown. Genetically Modified Babies Using CRISPR-Cas9.
Obstet Gynecol 2019.
OBSTETRICS & GYNECOLOGY
designed to cut the DNA on both sides of the unde-
sired sequence. However, cutting or disabling an
entire gene from a cell’s functional DNA may have
a negative effect on that organism, because many
genes code for more than one protein; genes and their
products may have unexpected interactions to other
genes and proteins, and genes may play different roles
when expressed in different parts of the body.
DR. JIANKUI HE
Dr. He received his PhD from Rice University in
2010, which was followed by postgraduate work at
Stanford University. After training in the United
States, Dr. He was recruited back to China as part
of China’s “Thousand Talents Plan” (www.1000plan.
org/en/young.html). Genetics is one of many areas of
interest to the Chinese government.15 It is rumored
that China will spend $9.2 billion on precision
(genetic) medicine over the next 15 years.16 In the
next year, Sichuan University’s West China Hospital
alone plans to sequence the genome of more than one
million people.16 Meghna Kataria, writing in BioN-
ews,17 reported that China has already treated 86 adult
patients with advanced cancer using CRISPR-Cas9
technologies. A description of at least 11 ongoing clin-
ical trials in China, involving treatment of cancer and
HIV, using CRISPR-Cas9, can be found in the U.S.
clinical trial registry (www.clinicaltrials.gov). These
trials go back as far as 2015 and have a projected
enrollment of more than 200 patients. Several Chi-
nese teams have previously edited human embryos
in experiments to eventually treat important diseases,
but without the intent of using them for human
implantation.18,19
Through conferences and travel, Dr. He main-
tained communication with many U.S. scientists
involved in genetics and genetic engineering. He
discussed his interest in modifying the genome of
embryos with several U.S. scientists, although, accord-
ing to news reports,20 these scientists did not believe
that he would proceed with this project at that time.
EMBRYO EDITING
Dr. He intended to create embryos that would be
resistant to most strains of HIV. The gene CCR5 co-
des for a protein that most HIV strains use to enter
cells.1 His objective was to eliminate production of
this protein in individuals born from the modified
embryos. He initially recruited eight couples in which
the father had HIV and the couple wished to inter-
vene so that their child would be prevented from ever
contracting HIV.
VOL. 134, NO. 1, JULY 2019 Rose and Brown
Dr. He’s approach was to delete a specific 32 base
pair segment of the CCR5 gene. This specifically tar-
geted deletion is called the CCR5 gene’s delta 32
mutation. It occurs naturally in about 10% of Euro-
peans.1 Creating a delta 32 mutation as opposed to
some other mutation, would be reassuring in that the
planned genetic alteration occurs naturally and there-
fore would be unlikely to cause unexpected adverse
health effects in the resulting children. However, it is
known that the delta 32 mutation is not entirely
benign. Having a nonfunctional CCR5 gene increases
a person’s susceptibility to West Nile virus and Japa-
nese encephalitis.21 It also may increase the likelihood
of an individual dying from influenza.22,23
One may question why Dr. He chose to modify
the CCR5 gene. Human immunodeficiency virus is
treatable, and the knowledge of how to decrease the
risk of contracting it is widespread. Perhaps the choice
to modify CCR5 gene was made because this modifi-
cation had been used successfully in somatic cells to
make CD4+ T cells resistant to HIV infection (using
zinc finger nuclease technology).24 Finding a worth-
while candidate gene to modify in germline cells may
be difficult. Established technology, namely preim-
plantation genetic testing, can be used to avoid most
identified mutations that cause recessive, X-linked, or
dominantly inherited diseases and still enable carriers
of these abnormal genes to have genetically related
children.25 Preimplantation genetic testing screens
embryos created with in vitro fertilization for the gene
of interest so that only nonaffected embryos are trans-
ferred back to the patient. At this early stage of devel-
opment, genetic editing of human embryos needs to
prevent diseases that cannot be prevented with exist-
ing technology.
In 2017, at a conference held at Cold Springs
Harbor Laboratory titled “Cancer and Stem Cells”
(available on YouTube), Dr. He presented research
on mouse, monkey, and human embryos. In this
research he looked at the problems of avoiding mosa-
icism and off-target DNA changes using CRISPR-
Cas9 on the CCR5 gene.
Reporters from The Wall Street Journal were able
to question Dr. He’s spokesperson after his talk
announcing this birth at the Hong Kong conference.26
Five women underwent in vitro fertilization, and
a total of 22 oocytes were retrieved. Each father’s
sperm was prepared in a manner to limit or eliminate
HIV in the sperm specimen. Reportedly, a sperm and
the CRISPR-Cas9/Cas9 enzymes were injected into
18 of the unfertilized oocytes. Thirteen edited
embryos were transferred back into the five women.
Two of the four embryos from one couple contained
Genetically Modified Babies Using CRISPR-Cas9 159
modifications of the CCR5 gene. According to Yong
reporting for The Atlantic,2 one of those transferred
embryos was known to still contain one copy of a nor-
mal CCR5 gene. This embryo transfer led to the birth
of Lulu and Nina. Lucas Laursen reported in Fortune
that there is at least one other pregnancy with a mod-
ified genome that is still ongoing.27
How to best perform gene editing of embryos
efficiently and avoid creating mosaic embryos while
modifying the genes inherited from both the mother
and father is a subject of active research. The first
Chinese team using CRISPR-Cas9 in 2015, and
editing genes of nonviable embryos, was able to
change the genome as desired in only 4 out of 86
embryos.19 More recently, a team at Oregon Health
and Science University edited genes of donated
embryos (without the intention of implanting them)
with markedly better results.28 The CRISPR-Cas9 en-
zymes were likely injected into zygotes as close to the
time of fertilization as possible.
Dr. He was successful in modifying the genes of
embryos that subsequently became babies. However,
it is troubling that he did not achieve what he set out
to do. At the Hong Kong conference where the births
were announced (and in the YouTube video that Dr.
He produced, dated November 25, 2018), he reported
that the genome of each child had been sequenced
and showed no changes other than in the CCR5 genes.
This suggests that this use of CRISPR-Cas9 made no
unintended changes in the embryo’s genome. How-
ever, because Lulu has one normal CCR5 gene that
will still produce the normal protein, she will not be
resistant to HIV infection. Of greater concern, accord-
ing to an (unnamed) expert who attended the confer-
ence where the births were announced, the slides
presented did not show a delta 32 mutation in any
of the CCR5 genes.2 The mutations that were created
in the CCR5 genes of Nina may or may not produce
functional proteins. If this report is accurate, the gene
modifications actually performed may not prevent Ni-
na from contracting HIV and may or may not have
other health consequences. In addition, even monitor-
ing the health of Lulu and Nina over their lifetimes
will not necessarily demonstrate that the objective of
preventing HIV infection was met, because they may
never be exposed to HIV.
Most countries regulate or ban research on
human embryos.29 China has guidelines for such
research. Investigators must present their research
proposal to the institutional review board for approval
at the institution where the research is to take place.
Dr. He was on the faculty of Shenzhen-based South-
ern University of Science and Technology. Yong re-
160 Rose and Brown Genetically Modified Babies Using CRISPR-Cas9
ported the University had turned down Dr. He’s
research proposal.2 The University subsequently is-
sued a statement condemning Dr. He’s experiment
and denying any knowledge of it (various news sour-
ces report that he was subsequently fired.) Dr. He had
taken a 3-year unpaid leave from the University start-
ing in February of 2018. He also worked at HarMon-
iCare Women and Children’s Hospital in Shenzhen,
which is independent of Southern University of Sci-
ence and Technology.26 Yong also reported that Dr.
He obtained institutional review board approval from
that hospital.2 The approval was published in the
China Clinical Trial Registry, but the hospital claims
that the signature on the approval form was forged.
As reported in the Atlantic, the Associated Press,
and The Wall Street Journal,2,20,26 much of the scientific
community was shocked at the risks intrinsic to this
undertaking. Although Dr. He achieved a “first”;
there are tangible risks involved in modifying
embryos at this time. Several studies have suggested
that off-target mutations after the use of CRISPR-Cas9
remain a significant problem and active area of
research.14,30–33 Gene editing in animals using both
CRISPR-Cas9 and prior techniques has demonstrated
occasional unintended consequences. For example,
there are a number of animal experiments of commer-
cial value in which an MSTN-like gene was deleted
(using CRISPR-Cas9 and prior tools). This gene codes
for myostatin, which limits how large muscles can
grow in humans.34 Animal gene editing experiments
to delete this gene had the intended effect of doubling
or tripling muscle mass, but genetically modified cat-
tle were created that were too big to exit though the
birth canal,35 some pigs were born with extra thoracic
vertebrae,7 and rabbits and pigs were born with
enlarged tongues.36 Gene function is difficult to fully
deduce and some applications of CRISPR-Cas9 result
in unintended genetic code changes with uncertain
effect.14,30–33
CONCLUSION
Dr. Jiankui He was the first scientist involved in the
birth of a baby with edited genes. He chose to edit
a gene related to a disease, HIV, which could both be
avoided and treated with established therapies. If the
recipient of the gene modification does not contract
HIV, it would not demonstrate the efficacy of this
gene modification because the individual may never
be exposed to HIV. He also chose to modify a gene to
eliminate a gene product that did not completely
protect the resulting child from the disease of concern.
The child could still be infected by strains of HIV that
used a different binding protein. Successfully
OBSTETRICS & GYNECOLOGY
eliminating this gene product by creating a delta 32
mutation, as planned, was known to create alternative
health issues for the recipient of the mutation. He
aided in the birth of a child who was heterozygotic for
a mutated CCR5 gene and who therefore would not be
immune to HIV infection. The other child born had
mutations of her CCR5 genes that might still produce
functional proteins, or as seen in animal experiments,
might present with unintended health outcomes.
More concisely, although the thought of gene
editing of embryos is an exciting prospect, our present
experience using gene editing for the treatment of
adults with severe disease or for beneficial genome
modification of animal populations is limited. Many
aspects of the experiment undertaken by Dr. He were
troubling. Even with the discovery of CRISPR-Cas9,
suboptimal control of molecular tools for gene editing
and a review of the history of gene editing suggest the
need for more caution and more collaboration before
undertaking additional attempts to modify germline
cells to create babies.
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PEER REVIEW HISTORY
Received January 22, 2019. Received in revised form April 1, 2019.
Accepted April 4, 2019. Peer reviews and author correspondence
are available at http://links.lww.com/AOG/B409.
OBSTETRICS & GYNECOLOGY