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Comment
Every Silver Lining has a Cloud: The Scientific and Animal
Welfare Issues Surrounding a New Approach to the
Production of Transgenic Animals
Robert D. Combes1 and Michael Balls2
1Independent
Consultant, Norwich, UK; 2c/o FRAME, Russell and Burch House, Nottingham, UK
Summary The scientific basis and advantages of using recently developed CRISPR/Cas-9 technology for transgenesis have been assessed with respect to other production methods, laboratory animal welfare, and the scientific relevance of transgenic models of human diseases in
general. As the new technology is straightforward, causes targeted DNA double strand breaks
and can result in homozygous changes in a single step, it is more accurate and more efficient
than other production methods and speeds up transgenesis. CRISPR/Cas-9 also obviates the use
of embryonic stem cells, and is being used to generate transgenic non-human primates (NHPs).
While the use of this method reduces the level of animal wastage resulting from the production
of each new strain, any long-term contribution to reduction will be offset by the overall increase
in the numbers of transgenic animals likely to result from its widespread usage. Likewise, the
contribution to refinement of using a more-precise technique, thereby minimising the occurrence of unwanted genetic effects, will be countered by a probable substantial increase in the
production of transgenic strains of increasingly sentient species. For ethical and welfare reasons,
we believe that the generation of transgenic NHPs should be allowed only in extremely exceptional circumstances. In addition, we present information, which, on both welfare and scientific
grounds, leads us to question the current policy of generating ever-more new transgenic models
in light of the general failure of many of them, after over two decades of ubiquitous use, to
result in significant advances in the understanding and treatment of many key human diseases.
Because this unsatisfactory situation is likely to be due to inherent, as well as possibly avoidable,
limitations in the transgenic approach to studying disease, which are briefly reviewed, it is
concluded that a thorough reappraisal of the rationale for using genetically-altered animals in
fundamental research and by the pharmaceutical industry, and for its support by funding
bodies, should be undertaken. In the meantime, the use of CRISPR/Cas-9 to generate new transgenic cells in culture is to be guardedly encouraged.
Key words: animal wastage, CRISPR/Cas-9, non-coding regions, non-human primates, nonpredictive disease models, reduction, targeted effects, transgenesis.
E-mail address for correspondence: robert_combes3@yahoo.co.uk
Introduction
The new approach referred to in the title is called
CRISPR/Cas-9, and was originally developed in 2012
(1, 2). The silver lining is the fact that it optimises,
simplifies, and shortens the time taken for producing
new transgenic animal strains. But the cloud, for
those concerned about animal welfare at least, is the
fact that its application will not only increase the
range and diversity of transgenic rodent strains, but
will greatly expedite transgenesis in other species,
including non-human primates (NHPs; 3).
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the expression of the transgene after its integration into the host cells. However, a key problem
with this production method is that several copies
of the transgene can integrate at random positions,
with the possibility that many of the founder
offspring will not express the right gene, or will
express it at inappropriate levels.
An alternative to pronuclear microinjection has
been the use of embryonic stem (ES) cells, in which
the transgene integrates at a few sites by low level
homologous recombination (5). However, considerable animal wastage still occurs, and in addition,
ES cells are not readily available for certain
species, such as NHPs. This was evidently a major
impetus for searching for new methods, culminating in the development of the CRISPR/Cas-9
methodology (2).
Comment
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of animals can also occur naturally during prenatal and post-natal development. Unfortunately,
there is a lack of published data on mortality rates
and ages at which deaths occur during the production of genetically-modified animals, and the use of
public databases detailing such information has
been called for (26).
A further cause of wastage arises as a result of
the need to remove variant animals exhibiting
inherent phenotypic instability, in order to maintain a high level of homogeneity in the colony (27).
In addition, commercial breeders might experience
variability in demand, to such an extent that they
have to maintain large stocks of animals, many of
which are unlikely to be sold (2830).
Previous attempts to address the low efficiency
of transgenesis include the injection of the transgene and appropriate regulatory sequences in the
form of an artificial chromosome vector (31), with
variable success. Other methods, particularly to
increase specificity for eliciting targeted DNA
changes, include gene-trap vectors, Cre-Lox recombination, TALENs (transcription activator-like
effector nucleases), and zinc finger nucleases. Like
CRISPR/Cas-9, the last-named technique induces
targeted double-strand breaks in DNA, and it has
been used to generate transgenic rats, as well as
transgenic mice (32). It is not the intention here to
describe these other methods in detail: suffice it to
say that each has its drawbacks, while the lastnamed three, in particular, have had variable
success in reducing the occurrence of random
insertion and other genetic effects.
Transgenic NHPs
It could be argued that, from a purely scientific
perspective, the area of transgenesis perceived to be
in most need of increased efficiency, is the production of transgenic NHPs, especially due to the high
financial costs of maintaining each animal, the long
time required for breeding, and problems in
acquiring the necessary ES cells. In the past, the use
of viral vectors has been the only way to generate
such animals. However, the CRISPR/Cas-9 method
has led to renewed interest in developing genetically-altered NHPs strains, as studies with them
have become financially and logistically more
manageable. One report has already been published
(33), demonstrating that such strains can be
produced by using the CRISPR/Cas-9 technique
instead of viral vectors. The genome-editing
complex, containing Cas-9 coding sequences and a
library of sgRNAs specific for three target genes,
were co-injected into one-cell-stage embryos of
cynomolgus monkeys. This yielded simultaneous
mutations in two of the target genes, as identified by
DNA sequencing. The latter technique was used to
verify the germline transmission of both of the trans-
Comment
genes in twins born after the transfer of geneticallymodified embryos into surrogate females.
The above data show that multiple mutations can
be induced in a single step in NHPs by using the
CRISPR/Cas-9 system. Moreover, no genetic alterations were detected in any off-target genomic sites,
confirming the high specificity of the process in
NHPs. However, these results should be regarded as
very preliminary. The apparent success of the
methods used, particularly the lack of unintended
genetic alterations, has yet to be confirmed.
Moreover, Niu et al. (33) commented that preliminary sequence data of both cultured embryos and
founder animals showed multiple genotypes, which,
they suggested, might be because nuclease cleavage
had occurred many times, at different stages of
embryogenesis. This issue would need to be resolved
by further genotyping. However, as Niu et al. themselves note, this will have to wait for the infant
founders to become older, to ensure the availability
of sufficient biopsy material. If this were to turn out
to be necessary for each new transgenic strain
produced in the same manner, it would increase the
time required and partially negate the advantages of
the CRISPR/Cas-9 technology. Also, any further
studies would be hampered by the five-month
gestation period.
Interestingly, a paper published very recently
(34), reports the first successful application of the
TALENs gene editing technique to produce transgenic NHPs the species in question being a
female cynomolgus monkey born with mutations
targeted to her methyl-CpG binding protein 2 gene
(MECP2), following the failure of several pregnancies. Although the founder animal is now several
months old, the same caveats apply to transgenesis effected by both this and the CRISPR/Cas-9
methods. In reality, there are few published data
for either method on the numbers of animals or the
time required. The CRISPR/Cas-9 study used 11
monkeys as egg donors, and 29 acted as surrogate
recipients, each one being injected with three eggs
(giving a total of 87 micro-injected embryos). This
compares with a corresponding figure of 200 eggs
which were needed to create ANDi, the first transgenic monkey (35). However, assuming that there
was confirmation of the preliminary analyses, of
the founders arising in both studies, it is possible
that many of the welfare problems and concerns
inherent in the two techniques, arising as a consequence of performing procedures on the animals,
as raised by Bottrill (35) with regard to the production of ANDi, would have been, at best, overcome,
or, at worst, alleviated.
Comment
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for this drug target in animal models were promising, these results have yet to be translated to the
clinic (52).
A similar story applies to Huntingtons disease
(53), seizures and epilepsy (54), as well as
Parkinsons disease (55), the transgenic animal
models for which have been criticised elsewhere
(56). Indeed, any progress that has been made, has
often been based more on the use of conventional
animal models, and studies involving patients (3).
Also, in a scientific appraisal of transgenic mouse
models of human disease, published eight years
ago, it was concluded that many such systems have
limited scientific relevance, and even fewer have
positively contributed to the development of novel
human medicines. Sadly, little seems to have
changed in the interim period, and the use of
transgenic animals clearly has a long way to go
before its claimed potential will, if ever, be even
remotely realised.
The reasons why a candidate drug, that shows
positive indications in a transgenic animal model
of the respective disease, fails to exhibit similar
therapeutic activity against the same disease in
volunteers, include: an incorrect genetic background; lack of a human-specific cytoplasmic factor
necessary for drug activity; use of a model based on
an inappropriate target/mechanism; lack of the
required levels of biotransformation of a pro-drug
to an active form in the mouse, or drug inactivation
in humans; and lack of transport in humans to the
target site (a particular problem with neuro-active
compounds and the effects of species-specificity on
passage across the bloodbrain barrier). Some of
these issues are inherent problems in the use of
transgenic rodents that might be difficult to overcome, while others are due to problems of the low
fidelity of mouse models in general.
We have previously speculated that genetic
alteration of mice, by ENU mutagenesis, or via
transgenesis, could change regulatory (non-coding)
regions of DNA (42, 56). We also noted that,
without full genomic sequencing, the occurrence of
such alterations in regulatory genes would remain
cryptic, in the absence of the target coding genes
being controlled, because the former have no
phenotypic effects on their own. This means that it
is more difficult to detect the involvement of regulatory genes, compared with coding sequences, in
the aetiology of disease, thereby increasing the
chance that transgenic strains are produced with
inappropriate targets and genetic backgrounds, in
cases where changes in regulatory genes are actually responsible for a disease phenotype. In these
circumstances, it would be expected that use of
such a mouse model of the disease would not result
in successful target validation. On the other hand,
successful validation should occur with a transgenic strain in which the correct regulatory gene
and genetic background had been used.
Comment
Comment
Conclusions
While the new CRISPR/Cas-9 technology reduces
the level of animal wastage resulting from the
production of each new strain, any long-term
contribution to reduction will be offset by the
overall increase in the numbers of transgenic
animals likely to result from its widespread usage.
Likewise, the contribution to refinement of using a
more-precise technique, thereby minimising the
occurrence of unwanted genetic effects, will be
countered by a probable substantial increase in the
production of transgenic strains of increasingly
sentient species.
Therefore, we still believe that the ECVAM
workshop statement should be the default position
for any assessment of proposed work on developing
and using transgenic NHPs in general. In other
words, such assessments, like those conducted by
ERPs (ethical review processes) and IACUCs
(institutional animal care and use committees), as
well as other regulatory and legislative bodies,
should lead to project approval only in extremely
exceptional circumstances. This conclusion has
also been taken in light of what we perceive to be
the inadequate protection of NHPs from general
usage in laboratories, afforded, for example, by
Directive 2010/63/EU (63).
In the meantime, we urge that further consideration be given to monitoring and exploring in
greater detail, the welfare consequences for all
species of every available method of transgenesis,
and to the wide application of the most promising
of these, such as CRISPR/Cas-9, for increasing the
diversity of cell lines for use in in vitro studies.
In addition, for both animal welfare and scientific reasons, there needs to be an in-depth critical
assessment of the rationale for using transgenic
143
References
1.
Le Cong, F., Ran, A., Cox, D., Lin, S., Barretto, R.,
Habib, N., Hsu, P.D., Xuebing, X., Jiang, W.W.,
Marraffini, L. & Zhang, F. (2012). Multiplex
genome engineering using CRISPR/Cas systems.
Science, New York 319, 819823.
2. Massachusetts Institute of Technology (2013).
Editing genome with high precision: New method to
insert multiple genes in specific locations, delete defective genes. [ScienceDaily, 03.01.13]. Available at:
www.sciencedaily.com/releases/2013/01/130103143
205.htm (Accessed 21.03.14).
3. Ragan, I. (2014). New tools new implications for
the 3Rs. Available at: http://blog.nc3rs.org.uk/newtools-new-implications-for-the-3rs/?utm_source=
Newsletter&utm_medium=Email%20newsletter&
utm_campaign=February%20newsletter (Accessed
23.03.14).
4. Anderson, J.J. & Dowdy, C.R. (2005). Transgenic
Animals, 61pp. Worcester, MA, USA: Worcester Polytechnic Institute. Available at: http://www.wpi.edu/
Pubs/E-project/Available/E-project-031405-135846/
unrestricted/IQP.pdf (Accessed 22.03.14).
5. Cartwright, E.J. (ed.) (2009). Transgenesis Techniques Principles and Protocols. Methods in Molecular Biology Series, Vol. 561, 3rd edn, 335pp. New
York, NY, USA: Humana Press, Inc.
6. Niemi, M. (2013). How emerging targeted mutation
technologies could change the way we study human
genetics. Available at: http://www.genomesunzipped.
org/2013/12/how-emerging-targeted-mutationtechnologies-could-change-the-way-we-studyhuman-genetics.php (Accessed 23.03.14).
7. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M.,
Doudna, J.A. & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in
adaptive bacterial immunity. Science, New York
337, 816821.
8. Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott,
D.A. & Zhang, F. (2013). Genome engineering using
the CRISPR-Cas9 system. Nature Protocols 8,
22812308.
9. Hsu, P.D., Scott, D.A., Weinstein, J.A., Ran, F.A.,
Konermann, S., Agarwala, V., Li, Y., Fine, E.J., Wu,
X., Shalem, O., Cradick, T.J., Marraffini, L.A., Bao,
G. & Zhang, F. (2013). DNA targeting specificity of
RNA-guided Cas9 nucleases. Nature Biotechnology
31, 827832.
10. Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L.,
144
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Comment
Reyon, D., Joung, J.K. & Sander, J.D. (2013). Highfrequency off-target mutagenesis induced by
CRISPR-Cas nucleases in human cells. Nature
Biotechnology 31, 822826.
Carroll, D. (2013). Staying on target with CRISPRCas. Nature Biotechnology 31, 807809.
Shen, B., Zhang, J., Wu, H., Wang, J., Ma, K., Li,
Z., Zhang, X., Zhang, P. & Huang, X. (2013).
Generation of gene-modified mice via Cas9/RNAmediated gene targeting. Cell Research 23, 720
723.
Wang, H., Yang, C.S., Shivalila, M.M., Dawlaty,
A.W., Cheng, F., Zhang, X. & Jaenisch, R. (2013).
One-step generation of mice carrying mutations in
multiple genes by CRISPR/Cas-mediated genome
engineering. Cell 153, 910918.
Yang, H., Wang, H., Shivalila, C.S., Cheng, A.W.,
Shi, L. & Jaenisch, R. (2013). One-step generation
of mice carrying reporter and conditional alleles by
CRISPR/Cas-mediated genome engineering. Cell
154, 13701379.
Li, D., Qiu, Z., Shao, Y., Chen, Y., Guan, Y., Liu, M.,
Li, Y., Gao, N., Wang, L., Lu, X. & Zhao, Y. (2013).
Heritable gene targeting in the mouse and rat using
a CRISPR-Cas system. Nature Biotechnology 31,
681685.
Sebo, Z.L., Lee, H.B., Peng, Y. & Guo, Y. (2013). A
simplified and efficient germline-specific CRISPR/
Cas9 system for Drosophila genomic engineering.
Fly (Austin) 8, 5257.
Anon. (undated). LASA Position Paper: Transgenics, 4pp. Tamworth, UK: Laboratory Animal
Science Association. Available at: http://www.lasa.
co.uk/PDF/position_transgenics.pdf (Accessed 24.
03.14).
Anon. (1999). Genetic Engineering: Animal Welfare
and Ethics. A Discussion Paper from the Boyd
Group. Available at: http://www.boyd-group.demon.
co.uk/genmod.htm (Accessed 25.03.14).
Camara, D., Dimitrova, Ir., Doynova, M., Jachacz,
L., Kachakova, D., Kepka, M., Ould Isselmou, C.B.,
Vorniere, J.P. & Yungarva, Tsv. (2008). Transgenic
and Cloned Animals: Ethical Problems? 20pp.
Brussels, Belgium: EU Socrates Erasmus European Community. Available at: http://bioethics.
agrocampus-ouest.eu/infoglueDeliverLive/digital
Assets/57413_transgenic_r.pdf (Accessed 22.03.14).
Anon. (undated). Data obtained from Home Office
and National Archives websites. Available at:
http://nationalarchives.gov.uk/ and https://www.gov.
uk/government/publications/non-technicalsummaries-granted-during-2013-volume-1 (Accessed 10.02.14).
Anon. (2013). Seventh Report on the Statistics on the
Number of Animals Used for Experimental and
other Scientific Purposes in the Member States of the
European Union, 14pp. Brussels, Belgium: European Commission. Available at: http://eur-lex.
europa.eu/LexUriServ/LexUriServ.do?uri=COM:20
13:0859:FIN:EN:PDF (Accessed 24.03.14).
Taylor, K., Gordon, N., Langley, G. & Higgins, W.
(2008). Estimates for worldwide laboratory animal
use in 2005. ATLA 36, 327342.
Hudson-Shore, M. (2012). Statistics of scientific procedures on living animals 2011: Another increase in
experimentation, but is there a shift in emphasis?
ATLA 40, 211219.
Home Office (2013). Annual Statistics of Scientific
Procedures on Living Animals Great Britain 2012,
Comment
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
145
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
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