Although CRISPR–Cas9 technology has been very successful, it does have some limitations.
HDR-mediated genome editing to introduce precise genetic modifications remains a much
less efficient process than NHEJ-mediated gene disruption, though progress has been
made in this regard.11, 12, 13 Multi-kb replacements by the HDR pathways are
challenging and require selections and/or large population cell sorting.14 Consequently,
the major applications for the HDR pathways are the local replacement of key regions
within genes. The ability to conveniently introduce precise site-specific modifications of
unlimited size currently represents an unmet need. Thus, CRISPR–Cas9-mediated
modifications are largely limited to applications involving gene disruption, applications
where modified cells have a competitive advantage over wild-type cells, and/or
applications where low-efficiency genetic modification is sufficient to produce the desired
phenotype. A second limitation relates to the off-target effects of Cas9, which raise
concerns about inadvertently disrupting genes of physiological importance15, 16 or
causing chromosomal deletions, inversions, or translocations.17 One can address these
concerns with a number of strategies to increase CRISPR–Cas9 specificity and decrease off-
target activity, including (i) selecting sgRNAs with few off-target sites within the genome
using bioinformatics software,18 (ii) transiently delivering Cas9 mRNA or
ribonucleoproteins (RNPs),19 (iii) using sgRNA or Cas9 variants with reduced DNA binding
activity, such as truncated, 17–18 nt sgRNAs,20 high-fidelity Cas9 (Cas9-HF1),21 or
enhanced-specificity Cas9 (eCas9),22 and (iv) using multiple gRNAs together with paired
nickases23 or fokI fusions to inactivated Cas9 (dCas9).24 A third limitation arises from the
delivery requirement and the fact that the commonly used Cas9 from Streptococcus
pyogenes bacteria (SpCas9) is approximately 4 kb and hence is a rather large cargo for a
viral vector, such as adeno-associated virus (AAV). The MIT lab of Zhang and coworkers has
developed another variant of Cas9, derived from the Staphlococcus aureus bacteria
(SaCas9) that is 1 kb in length—substantially shorter than SpCas9, thus expediting delivery
with AAV. This group has reported that the SaCas9 exhibits an efficiency in genome editing
that is similar to that of SpCas9.25 In the past, most methods of genetic modification were
restricted to use in model organisms, such as the mouse, whose cells could be
manipulated via homologous recombination.1 CRISPR–Cas9 has widened the scope of
transgenic animal production by enabling facile genetic modification of embryonic stem
cells (ESCs), as well as direct modification of zygotes, which is applicable for most
species.26 CRISPR–Cas9 has been used productively in a wide variety of animals, including
nematodes,27 fruit flies,28 malarial mosquitoes,29 sea urchins,30 zebrafish,31 sea
anemone,32 mice,33 nonhuman primates (NHPs),34 and even human zygotes.35 These
examples will be discussed below.
Ethical considerations
There have been, and continue to be, important debates surrounding the ethical aspects
of gene editing technologies.105, 106 As CRISPR–Cas9 technology has accelerated the
ability to create transgenic animals, these discussions have become increasingly relevant.
Debates have been most intense regarding editing of the human genome, especially of the
germline, but have also concerned editing of nonhuman animal genomes. For example,
the proposal to use CRISPR–Cas9 gene drives to eradicate alleles
(Ellen Shrock, 2017)
Treatment of diseases
CRISPR-Cas9 can be applied to cells in vivo or ex vivo. In the in vivo approach, CRISPR-
Cas9 is directly transferred to cells in the body using either viral or nonviral methods. In
the ex vivo approach, first the cells are removed from the body; then CRISPR is applied
to the cells and they are transferred back to the body (Roh et al., 2018). This approach
has great potential to develop tissue-based therapies (Rath et al., 2015). Using CRISPR-
Cas9, the mutation in the dystrophin protein responsible for the most common form of
Duchenne muscular dystrophy was successfully removed (Amoasii et al., 2018; Duchêne
et al., 2018; Koo et al., 2018; Long et al., 2018). There are studies to prevent and treat
AIDS by inhibiting the entry of HIV into the cell or by removing the HIV genome
integrated into the host genome using CRISPR-Cas9 (Saayman et al., 2015). Induced
pluripotent stem cells (iPSCs) were successfully produced from cystic fibrosis patients
with confirmed F508 deletion in the cystic fibrosis transmembrane regulator (CFTR) gene
by CRISPR-Cas9 (Firth et al., 2015). There are also studies for cataracts (Wu et al., 2015;
Yang et al., 2016) and Parkinson’s disease (Yang et al., 2016). However, recent studies
have shown that CRISPR-Cas9 activates the type 1 interferon (INF) pathway, causing a
type 1 INF-mediated immune response (Kim et al., 2018; Charleswort et al., 2019). These
findings currently limit the use of CRISPR-Cas9 in treatment.
Ecological imbalance
In studies using RNA-targeted gene editing methods based on CRISPR-Cas9, nontarget
effects should be examined in depth. Since gene drift will persist in a population,
possible off-target mutations will continue in each generation. In addition, the number
and effect of mutations may increase as generations progress (Rodriguez, 2016;
Hundleby and Harwood, 2019). Another concern is the possibility that genes can be
transferred to other species in the environment. Transferring the regulated sequences to
other species may result in the transmission of negative characteristics to the associated
organisms (Esvelt et al., 2014). The distribution of the properties of the entrained genes
among the populations can make control very difficult.
Generation of chimeric animals for organ transplantation
Organ transplantation is the replacement of an organ that cannot function in an
individual’s body with a healthy organ from a living donor or cadaver. The primary
purpose is to save the life of the patient, who is in danger of organ failure, and to
increase the lifespan and quality of life (Black et al., 2018). The development of chimeric
animals may prevent patients from spending precious time waiting for an appropriate
donor.
Bioethical issues in the generation of chimeric animals arise from the fact that chimeras
contain human nerve and germ cells (Polcz and Lewis, 2016). The two main issues can be
summarized as defining the order of nature and the moral disorders caused by how the
organism is treated depending on whether the organism is accepted as human or
animal. Some people think that chimeric embryos will affect human dignity and identity
because they have the power to develop organisms with human-derived cells and
tissues. The others state that chimeric organisms containing human cells cannot turn
into humans and therefore will not affect human dignity. They also argue that the
human-like features imparted to chimeras will neither affect the biological environment
nor the moral status of animals and will never reach human consciousness (Koplin,
2019; Degrazia, 2019).
Animal welfare and dignity
Animal welfare is another bioethical concern encountered during the application of
genome editing technologies on animals. First of all, the possibility of off-target
mutations in the genome can lead to diseases or different side effects in animals (Ishii,
2017a; Schultz-Bergin, 2018; de Graeff et al., 2019). Such a situation will adversely affect
animal welfare (Rodriguez, 2017).
The second bioethical issue to be discussed could be the concerns about “animal
dignity” (Eriksson et al., 2018) and alterations in their natural environments and
physiological needs (Manesh et al., 2014). Some studies have stated that the use of
animals as objects only serving for humans is not ethically or morally acceptable
(Martinelli et al., 2014; Fung and Kerridge, 2016; Greenfield, 2017), and such practices
can lead to greater control over humans on animals (Ishii, 2017a; de Graeff et al., 2019).
Some others think that animals are not bound by any moral law and therefore there is
no need for a discussion regarding animal dignity (Heeger, 2015; Shriver and
McConnachie, 2018). Schultz-Bergin (2017) stated that animal rights, welfare, and
dignity will not be adversely affected since these animals will occur through genome
editing technologies. The existence of contrary opinions on this matter indicates that the
mentioned bioethical issues will be on the agenda for a long time.
There are serious concerns about changes in the natural ecosystem that may occur if the
GMOs produced with CRISPR-Cas9 are released to the ecosystem in a controlled or
uncontrolled manner. Considering the applications of CRISPR-Cas9 that protect
mosquitoes from malaria parasites (Gantz et al., 2015) or make female mosquitoes
infertile (Hammond et al., 2016), the effect of GM mosquitoes on other organisms with
which they are associated in their ecosystems cannot be predicted. It is clear that small-
scale research in the laboratory does not fully reflect possible changes in the natural
ecosystem (Carroll, 2017). In agriculture, another concern about GMOs produced with
CRISPR-Cas9 is whether they will be accepted by the public. GMOs that were produced
using different technologies in the past faced harsh public reactions (Carroll, 2017).
Furthermore, the fact that GMOs produced with CRISPR-Cas9 are difficult to identify
outside the laboratory raises safety concerns (Shinwari et al., 2017). Before the launch
of such products, the necessary explanations and declarations should be made by the
authorities in a transparent and clear manner in order to prevent misjudgments and
questions that may occur in the public, and precautions and arrangements should be
established to ensure the safety of the public.
Fatma, 2020
CRISPR—Limitations
There are potential limitations associated with the use of CRISPR–Cas9
technology. In research using RNA-targeted gene editing technology based on
CIRSPR-Cas9, all off-target effects should be carefully investigated. The
consequence of the persistence of genetic drift in a population could be the off-
target mutations that will persist in each generation. Moreover, effect and
number of mutations can increase as the generations progress [110]. The
dispersion of a feature of gene driving can be difficult to control. What is more,
destroying the whole population targeted by gene drive can have severe effects
in the ecosystem’s balance. Another issue is the potential transfer of genes to
other species in the environment. The consequence of this can be the
transmission of negative features to related organisms [111,112]. By comparison,
off-target mutations are more common in human cells than in zebrafish or mice
[113,114]. Genetic mosaicism in founders can be one of the results of CRISPR–
Cas9 mediated gene editing in embryos, especially in the generation of
transgenic and knockout animal models. CRISPR–Cas9 components are usually
injected as RNA, DNA or protein molecules directly to fertilized zygotes
[114,115,116]. This is undesirable in the majority of applications due to the
formation of false-positive genotyping results. For example, the founder mouse
can show a homozygous deletion of candidate based on tail DNA genotyping;
however, it may never transmit the deletion allele to the offspring [117]. Genetic
mosaicism makes phenotype analysis difficult in F0 animals, especially when
carrying-out HDR-mediated targeted DNA insertions. Many F0 mosaic embryos
should be screened to receive homogenous germline transmissions with the
desired mutations [118,119]. Tu et al. have reported that tagging Cas9 with
ubiquitin-proteasomal degradation signals can simplify the Cas9 degradation,
thereby reducing mosaic mutations and increasing its ability of genome
modification in non-human primate embryos [120].
15. CRISPR—Ethical Considerations
Significant advances in CRISPR–Cas9 technology have introduced challenges in
regulating its safe and ethical use. Several groups of scientists have used CRISPR–
Cas9 to edit genes in human embryos, even though it was always done in non-
viable, triploid zygotes [121,122]. This type of research was carried out to
investigate the specificity and accuracy of the CRISPR–Cas9 system [123]. This led
to a debate in the scientific community on how to ethically and responsibly use
gene editing methods in a way that does not hinder the benefits of research
results and discoveries [124]. In November 2018, the U.S. National Academy of
Sciences, the U.S. National Academy of Medicine, the Royal Society of the United
Kingdom and the Academy of Sciences of Hong Kong convened the Second
International Summit on Human Genome Editing. Over 500 researchers,
policymakers, ethicists, representatives from medical and scientific academies,
patient groups’ representatives and others attended the summit. During the
event, the potential benefits and risks of editing the human genome; cultural
and ethical perspectives; regulatory and policy issues; and public outreach were
debated. The Second International Summit followed the First International
Summit on Human Genome Editing in December 2015. In a statement published
after a previous summit, the committee noted that intensive basic and preclinical
study on genome editing was necessary and this type of study should be subject
to proper legal and ethical rules concerning both somatic (nonheritable) and
germline (heritable) human genome editing. Despite the fact of many
precedents, the general acceptance of modifying single person’s somatic DNA is
more tolerable than a germline, which could be passed to offspring. The first case
is significantly beneficial in treatment of hemophilia, HIV, cancer, Alzheimer’s
disease and any other novel diseases [125,126]. In the second case, germline
gene modification lacks societal consensus, and some countries even outlaw this
practice. What is more, genome editing of germline cells that could be passed on
to the next generations as a part of the human gene pool seems irresponsible
until the safety concerns are resolved. It should be mentioned that this aspect
should consider the prevention of the transmission of genetic variants associated
with illness, decreasing the probability of diseases developing and enhancing
human capabilities. In first regard, genome editing will give a parents another
option instead to pass to offspring the genetic variants responsible for, e.g.,
Huntington’s disease or not having biologically related children at all. The second
option, will allow one to inactivate the particular genes resulting in lowering
blood pressure or cholesterol level. The third and the most controversial, will
allow one to enhance human functions and create superhumans by increasing,
e.g., strength [73]. At the second summit, it was found that germline genome
editing could be morally permitted in certain circumstances, but there are no
such circumstances in the world. In addition, one opinion was that the benefits
and risks of the genome editing of germline cells are still not clear enough to
allow germline genome editing to continue
(Janik, 2020)
Although the mosaicism is an undesired result in genome editing
animals, there may be several advantages, which include enabling
animals to survive beyond the lethal phase when manipulated genes
are essential to animals. In addition, mosaic animals help us better
understand dosage effects of genes on developmental defects,
especially those which may mimic human congenital disorders (Zhong
et al., 2015). However, the mosaicism caused by CRISPR/Cas9 is
undesirable in most cases. The biggest problem of chimeric large
animals is that the mutation may be difficult to be transmitted into the
offspring (Oliver et al., 2015) because of the long breeding cycle of
large animals especially nonhuman primates. There are several
possible strategies to reduce the mosaicism. The first way is to speed
up the editing process by the introduction of CRISPR/Cas9 components
in an appropriate format [Cas9/sgRNA ribonucleoprotein (RNP)] and
concentration into very early pronuclear stage zygotes by using
electroporation (Mehravar et al., 2019), so the CRISPR/Cas9 system will
work at the earliest stage of zygotes. Secondly, as described above,
shortening the longevity of Cas9 in combination with embryo splitting
to eliminate the delayed function of Cas9 also makes a difference (Tu
et al., 2017). The third strategy is to use germline modification. In this
way, genetically-modified somatic cells can be used as nuclear donors
for SCNT into enucleated germ cells. In another way, generally targeted
gene edited spermatogonial stem cells (SSCs) can be used as donors
for transplantation into testis directly.
The last problem is the efficiency of the CRISPR/Cas9 system.
According to the previous study, the efficiency of gene targeting with
CRISPR in large animals (like nonhuman primates) is more variable and
lower than that in mice (Chen et al., 2016). Therefore, precise gene
editing technologies need to be further improved, to increase the
efficiency of gene targeting and the rate of homozygous mutation by
using new Cas protein [like Cpf1 (Cas12a)] and new systems (like base
editing system).
Even so, there are still many limitations in the establishment of large
animal models, which may involve the inadequate gene targeting
efficiency, mosaicism, and off-targeting. Many strategies and optimized
components of the system have been brought up to reduce these
drawbacks or to improve efficiency.
(LIN, 2022)
Clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated enzyme (Cas) is a naturally
occurring genome editing tool adopted from the prokaryotic adaptive
immune defense system. Currently, CRISPR/Cas9-based genome
editing has been becoming one of the most promising tools for
treating human genetic diseases, including cardiovascular diseases,
neuro-disorders, and cancers. As the quick modification of the
CRISPR/Cas9 system, including delivery system, CRISPR/Cas9-based
gene therapy has been extensively studied in preclinic and clinic
treatments. CRISPR/Cas genome editing is also a robust tool to create
animal genetic models for studying and treating human genetic
disorders, particularly diseases associated with point mutations.
However, significant challenges also remain before CRISPR/Cas
technology can be routinely employed in the clinic for treating
different genetic diseases, which include toxicity and immune response
of treated cells to CRISPR/Cas component, highly throughput delivery
method, and potential off-target impact. The off-target effect is one of
the major concerns for CRISPR/Cas9 gene therapy, more research
should be focused on limiting this impact by designing high specific
gRNAs and using high specificity of Cas enzymes. Modifying the
CRISPR/Cas9 delivery method not only targets a specific tissue/cell but
also potentially limits the off-target impact.
(Baohong Zhang, 2020)
