A new gene-editing technique has taken the world by storm, it
can disable or change specific gene in the genome of all animals including
humans faster and more efficiently than ever before but it has raise
unprecedented concern over safety and ethicsDr Mae-Wan Ho
The new gene-editing technique CRISPR (see Box)  has
taken the world by storm. It enables geneticists to disable or change the
sequence of specific genes in the genome of practically all animals including
humans faster, more efficiently than ever before, promising to improve our
understanding of how genes work, delete genes that cause diseases, even modify
human embryos to rid them of diseases or to ‘enhance ‘ them. The applications
are moving ahead so fast that many scientists are calling for caution as major
safety and ethical concerns need to be addressed.
The issue came to a head when a team of
Chinese researchers created the first genetically modified human embryos using
the new wonder tool for genetic modification
(clustered regularly interspaced short palindromic repeat) refer to short,
partially repeated DNA found in the genome of bacteria and other microorganisms
that protect the organism against viruses (see Figure 1).
Figure 1 CRISPR-mediated immunity (see text)
Fig. 1, CRISPR regions are composed of short DNA repeats (black diamonds) and
spacers (colour boxes). When a new virus infects the bacterium, a new spacer
derived from the virus is incorporated among the existing spacers. The CRISPR
sequence is transcribed and processed to generate short CRISPR RNA molecules.
The CRISPR RNA associates with and guides bacterial DNA cutting protein (Cas9
protein) to a matching target sequence in the invading virus. The Cas9 protein
cuts up and destroys the invading viral genome.
has become the latest gene-editing technique, which enables precise changes to
be made in the genes of fruit flies, fish, mice, plants and human cells. To do
that, geneticist first design and synthesize short a RNA molecule that match a
specific DNA sequence. Then, as in the targeting step of the bacterial system,
this guide RNA shuttles the Cas9 protein to the intended DNA target, and can
silence a gene or change the sequence of a gene by adding a repair template
with a specified change in sequence, so that it is incorporated into the DNA
during the repair process. The targeted DNA is now altered to carry the new
sequence (see Figure 2).
Figure 2 CRISPR gene silencing or gene editing
First genetically modified human embryos created in China
A team of researchers at Sun Yat-sen University in
Guangzhou China used CRISPR to edit the b-haemoglobin gene (HBB) in human pre-implantation embryos .
Actually the team used defective embryos with three pronuclei that normally
would not be implanted. They found that CRISPR/Cas9 could effectively cut the HBB gene, but the efficiency of homologous
recombination directed repair of HBB was low and the edited embryos were
mosaic. Off-target cleavage was also found. Moreover, the endogenous d-haemoglobin gene (HBD), which is homologous to HBB,
competed with exogenous donor sequence to act as the repair template, resulting
in untoward mutations. The researchers concluded: “Taken together, our work
highlights the pressing need to further improve the fidelity and specificity of
the CRISPR/Cas9 platform, a prerequisite for any clinical applications of
Even before the official publication of the
paper, two independent groups of scientists wrote editorials in the journals Nature
 and Science  respectively expressing their concerns. The
group in Nature  called for a halt to editing the human germ line on
grounds that heritable human genetic modifications pose serious risks,
and the therapeutic benefits are tenuous. It would also jeopardize current
efforts to apply the technology to somatic cells as a potential functional cure
for HIV/AIDs and b-thalassaemia.
Studies using gene-editing in animals such as rats, cattle, sheep and pigs show
that it is possible to delete or disable genes in an embryo – a simpler process
than actually correcting or changing the DNA sequence – in only some of the
cells. The precise effects of genetic modification of an embryo may not be known
until after birth, and long after birth. Patient safety is the primary concern,
before ethical concerns are considered. Today some 40 countries ban it. Fifteen
of 22 nations in Europe prohibit the modification of the germ line. The US
NIH’s Recombinant DNA Advisory Committee explicitly states that it “will not at
present entertain proposals for germ line alterations.” Non-therapeutic genetic
enhancement is also a concern.
The group in Science  emphasize
that research is needed to understand and manage risks arising from the use of
the CRISPR-Cas9 technology. Considerations include the possibility of
off-target alterations, as well as on-target events that have unintended effects.
It is critical to implement standardized benchmarking methods to determine the
frequency of off-target effects and to assess the physiology of cells and
tissues that have undergone genome editing. The potential safety and efficacy
issues arising from the use of this technology must be thoroughly investigated
and understood before any attempts at human engineering are sanctioned.
before an International summit in
December 2015 co-sponsored by the US National Academy of Sciences, the US
National Academy of Medicine, the UK Royal Society and the Chinese Academy of
Sciences to consider the scientific and social implications of genome editing, Jennifer Doudna, a researcher at University of California
Berkeley who helped invent CRISPR/Cas9, and a
signatory on the editorial in Science, wrote in a commentary in Nature
 stating that “we do not yet know enough about the capabilities
and limits of the new technologies, especially when it comes to creating heritable
mutations.” Hence, “human-germline editing for the
purposes of creating genome-modified humans should not proceed at this time,
partly because of the unknown social consequences, but also because the
technology and our knowledge of the human genome are simply not ready to do so
safely.” She also stated that future discussions should
address other potentially harmful applications of genome editing in non-human
system, such as “the alteration of insect DNA to ‘drive’ certain genes into a
population” (see below).
In the event, the
international summit called for a slowdown on research
involving heritable modifications of the human genome . Although the academies
acknowledged that such research has the potential to eradicate genetic diseases
or enhance human capabilities, they also said the science is just too new to do
any of that safely or successfully.
they argued it is alright to use germline cells or early human embryos in basic
and preclinical lab research, so long as they are not then used “to establish a
pregnancy”. (This goes beyond what Francis Collins, National Institutes
of Health's director, said on the publication of the China experiment, that the
NIH would not fund genomic editing involving human embryos, even if the embryos
were not used to create a pregnancy.)
Not all scientists are satisfied with the
outcome of the summit . Paul Knoepfler a cell biologist at University of
California Davis said he was disappointed that the organisers did not propose
at least a temporary moratorium on germline human genetic modification.”
January 2016, a team led by Keith Joung at Harvard Medical School, Boston,
Massachusetts in the United States reported the successful construction of a
high-fidelity CRISPR/Cas9 nuclease that has little or no detectable genome-wide
off-target effects. This is achieved by changing 4 amino acids with long side
chains to one with a short side chain (alanine) to reduce the protein’s
non-specific interaction with the phosphate DNA backbone. The resultant
SpCas9-HF1 (Streptomyces pyogenes Cas9-high-fidelity 1) nuclease retains
on-target activities comparable to the wild type enzyme with 85 % of the single
guide-RNAS (sg-RNA) tested in human cells, with very little or no off-target
effects. The specificity of SpCas9-HF1 and its potential target was improved
with further substitutions to reduce non-specific interactions with the DNA .
Reducing off-target effects does contribute
substantially to safety. But other safety aspects such as unintended
consequences from on-target events and the stability of the modifications, as
well as ethical concerns still need to be addressed.
In addition, other worrying applications
raise new issues on safety, such as ‘gene drive’ in transgenic insect disease
‘Gene drive’ for transgenic mosquitoes
Health Organization reports that mortality from malaria continues to decrease
and estimates that ~3.3 million lives have been saved since 2001 as a result of
new drugs, personal protection, environmental modification and other measures;
but there were still ~580 000 deaths globally from malaria in 2014 .
at Universities of California Irvine and San Diego has just created a
transgenic mosquito in the malaria vector species Anopheles stephensi
carrying genes against the malarial pathogen Plasmodium falciparum using
CRISPR/Cas9 in a new construct to produce a ‘gene drive’ that makes almost all
the progeny of the transgenic male mosquitoes anti-Plasmodium .
is actually a mutagenic chain reaction first devised by two of the researchers at
UC Irvine in Drosophila melanogaster . Previous gene editing was
done with Cas9 and the guide RNA and transgene sequence on separate
plasmids. By engineering Cas9 next to the gRNA and transgene sequences in a
single plasmid, a mutagenic chain reaction is triggered that converts the
unmodified gene in the wild-type chromosome to mutant state.
stephensi is estimated to be responsible for ~12 % of all transmission in
India, mostly in urban environments, accounting for ~106 000 clinical cases in
2014 . Laboratory strains are transformed efficiently with transposable
elements to facilitate analysis of transgene expression in diverse genomic
locations. Site-specific integration allows insertion of exogenous DNA into the
mosquito genome at locations with minimum impact on fitness . Furthermore,
a dual anti-parasite gene was developed based on the single-chain antibodies
miC3 and m2A10 that target the Plasmodium ookinete protein Chitinase 1
and the cricumsporozoite protein respectively . Transgenic female mosquitoes
expressing miC3 and m2A10 were free from P. falciparum sporozoites
(infectious stage of the parasite) in their salivary glands under infection
conditions expected in the field, and hence incapable of transmitting the
parasite. Modeling of gene drive system, which exceed Mendelian inheritance,
results in a more rapid transformation of a population with fewer releases than
the ‘inundative’ approach, in which engineered mosquitoes (without gene drive)
ware released in numbers substantially exceeding those of the local populations
considerations encouraged the researchers to construct their gene-drive system in
A. stephensi using CRISPR/Cas9-mediated homology-directed repair (HDR)
adapted from the mutagenic chain reaction developed in the fruit fly. The drive system as designed works in both the
male and female germ lines of mosquitoes derived from transgenic males .
Cas9-mediated gene targeting is also evident in the somatic cells of embryos
derived from transgenic females. The system can carry
a relatively large set of genes (~17kb in length).
The structure of the gene-drive plasmid
consists of the Cas9 gene and its promoter, the gRNA and its promoter, the dual
antibodies genes with their promoters and marker gene encoding the Drosophila
red fluorescent protein (DsRed) with its promoter; this long continuous stretch
flanked by sequences homologous to the target the autosomal gene kh encoding
the enzyme kynurenine hydroxylase. Altogether, the plasmid is 21 kb in length
with 16 625 bp comprising the ‘cargo’ to be inserted into the target gene.
A total of 680 G0 wild-type
embryos were injected with the transforming plasmid and other components to aid
transformation; 122 and 129 males and females respectively survived to the
adult stage, and were assigned to 22 male-founder and 9 female-founder pools
and outcrossed to wild type adults of the opposite sex. Two males positive for DsRed,
designated 10.1 and 10.2 were recovered after screening 25 723 G1 larvae.
When outcrossed to wild-type females, 10.1
produced all DsRed+ adult progeny (n=14), whereas 10.2 produced only
57 of 129 (44 %) DSRed+ adult progeny. DSRed+G2
males and females from both 10.1 and 10.2 were outcrossed to wild-type mosquitoes, and the G3
larvae progeny were scored for DSRed. Line 10.1 produced 1 321 (99.7 %) DSRed+
while line 10.2 produced 4 631 (99.2 %) DSRed+ G3 larvae.
These results are consistent with highly efficient gene-drive.
When the 10.1 and 10.2 G3 males
and females were outcrossed to wild type mosquitoes of the opposite sex, the G4
male and female progeny of G3 males and females derived from G2 10.1
and 10.2 transgenic males show a high frequency of DsRed transmission,
corresponding to a 96.9 % rate of gene conversion. In contrast, a much high
proportion of G4 larvae progeny of G3 males and females
derived from G2 10.2 and 10.2 transgenic females appeared to
have inherited mutations at the kh locus instead of gene-conversion
events with a ratio of 1.33 DsRed+ to 1.0 DsRed-. This
indicates that the transgene is unstable in the female, and there as key safety
issues involved (see later).
Importantly, the anti-Plasmodium genes
are actively transcribed in the DsRed+ mosquitoes.
Gene-drive under fire
Long before the transgenic mosquito with gene drive was constructed,
a group of scientists have expressed their concerns as the ‘regulatory gap’
regarding such insects . They highlighted the need for containment
measures and ‘reverse drive’ to undo the process. They point out that gene
drive has not been evaluated for safety. A drive may move through only part of
a population before a mutation inactivates the engineered trait. (This was
indeed the case in transgenic female mosquito progeny, where the CRISPR/Cas is
also active in somatic cells, see above). In some cases preferred phenotypes
might be maintained as long as new drive encoding updates are periodically
also made the key point that (p. 627): “In theory, precision drives could limit
alteration to target populations, but the reliability of these methods in
preventing spread to nontarget or related populations will require assessment.
To what extent and over what period of time might crossbreeding or lateral
[i.e., horizontal] gene transfer allow a drive to move beyond target populations?
Might it subsequently evolve to regain drive capabilities in populations not
This is crucial in the light of the
instability of the gene drive in transgenic female mosquitoes reported .
When these females bite animals including humans, there is indeed the
possibility of horizontal gene transfer of parts, or the entire gene-drive
construct, with potentially serious effects on animal and human health. Cas9
nuclease could insert randomly or otherwise into the host genome, causing
insertion mutagenesis that could trigger cancer or activate dominant viruses.
In addition, the many transcripts and gene products encoded by the gene-drive
construct could also have harmful effects including immunological reactions. These
same hazards apply, all the more so to species of animals that feed on
mosquitoes, as it is now well-known that nucleic acids in food can get into
cells and tissues (see  Nucleic Acid
Invaders from Food Confirmed, SiS 63).
the ecological risks of gene drives are enormous, so warns conservation
scientists from Australia’s Commonwealth Scientific and Industrial Research
Organisation . They stated: “The question is no longer whether we can
control invasive species using gene drive, but whether we should.” As the gene
drive can in principle lead to the extinction of a species, this could involve
the species in its native habitat as well as where it is considered invasive.
As distinct from conventional biological control, which can be applied locally,
there is no way to control gene flow. They point out that because the
CRISPR/Cas gene drive remains fully functional in the mutated strain after it
is created, the chance of off-target mutations also remain and the likelihood
increases with every generation. “If there is any risk of gene flow between the
target species and other species, then there is also a risk that the modified
sequence could be transferred and the adverse trait manifested in nontarget
organisms.” (This commentary has not even begun to consider horizontal gene
flow, which would multiply the risks many-fold.)
is also increasing awareness that many invasive species will have considerable niche overlap, such that removal of one species will
enable another to rapidly take its place.
They call for a
thorough ecological risk assessment before any application of CRISPR/Cas gene
drive is contemplated in the control of alien species, to prevent a ‘silver
bullet’ becoming a ‘conservation threat’.
There are numerous reasons to proceed with caution with
CRISPR/Cas9 applications. It is a powerful, efficient, and cheap gene-editing
tool beset with risks for health and the environment. Particularly worrying is
its use in gene-drive, a currently irreversible and uncontrollable process once
released into the environment.
This account is modified from “CRISPR: a
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NT, Zheng Z and Joung JK. High-fidelity CRISPR-Cas9 nucleases with no
detectable genome-wide off-target effects. Nature 2016, doi:10.1038/nature16526
World Health Organization (2015) World Malaria Report, 2014 (WHO, Geneva, Switzerland/
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Todd Millions Comment left 13th January 2016 14:02:53 I'm confused! According to the our Sages of P.R. science-we already have such exquisite precision in our gene engineering.With NO off target effects and utter stability and predictability!This MUST be true as all our holy bureaucrats and sacred administrators SAY so(except for the heretics). So why do we need a new one? Has something gone wrong?
Rory Short Comment left 15th January 2016 12:12:37 This is exciting new technology but it is new therefore we should proceed with the utmost caution and keep it firmly in the lab for now.