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European Biopharmaceutical Review
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Last year’s discovery of a revolutionary gene editing tool created huge
excitement in the biomedical industry. The CRISPR/Cas9 system – which
enables the targeted modification of DNA in living organisms – offers
greater efficiency, precision and flexibility than previous gene editing
technologies – and with it, the potential to eradicate numerous
diseases
There was once a time where gene editing – or genome
engineering – was considered to be science fiction, due to its long and
random process: it started with the deletion or insertion of a DNA
sequence at a specific location, and increased its accuracy through a
technique called homologous recombination. Because the desired
recombination events occur very infrequently (1 in 106-109 cells), using
this method presented enormous challenges to the large-scale
application of gene-targeting experiments and, as a result, its
application has been limited in most organisms (1,2).
DNA-Binding Proteins
Today,
genome editing is based on the use of engineered or programmable
nucleases composed of sequence-specific DNA-binding domains bound to a
non-specific DNA cleavage module (3). These nucleases are guided to a
specific sequence within the genome to induce a double-strand DNA break
(DSB). When a DSB is generated, the cell’s intrinsic DNA repair system
is activated and the genome is modified during the repair of the DSB.
DSBs are typically restored by either non-homologous end joining or
homology-directed repair (2). There are three main DNA-binding proteins
that have been engineered thus far; however, each of these carry
challenges with either specificity, flexibility or adaptability of gene
editing (1):
● Meganucleases derived from microbial mobile genetic elements
● Zinc-finger nucleases based on eukaryotic transcription factors
● Transcription activator-like effector nucleases from Xanthomonas bacteria
Following
the discovery of the clustered regularly interspaced short palindromic
repeats (CRISPR) endonucleases-RNA-guided DNA endonuclease (Cas9) from
the type 2 bacterial adaptive immune system, everything has changed and
gene editing has become a lot easier.
System of Choice
In
the CRISPR/Cas9 system, the protospacer-adjacent motif (PAM) is the
critical element, located at the 3' end of the DNA target site, and
dictating the search mechanism for the DNA target with Cas9 (1). Several
studies have demonstrated that PAM is involved in the binding of the
Cas9 to the target and the DSB (1). Target sequences without PAM do not
induce DSB (4). Cas9 (formerly known as Cas5, Csn1, or Csx12) is the
only enzyme within the Cas gene cluster that facilitates target DNA
cleavage (1).
The CRISPR/Cas9, type 2 system is emerging as the
sequencespecific nucleases of choice for genome engineering for three
reasons (2):
1. Cas9 is guided by a single-guide RNA (gRNA) that
is easily engineered. The gRNA targeting sequence consists of 20
nucleotides (nt), which is homologous to the DNA target site and can be
ordered as a pair of oligonucleotides and rapidly cloned
2. The
modular features of the CRISPR/Cas9 system and short 20nt length of the
targeting gRNA makes these components advantageous in being able to
target and cleave multiple target sequences simultaneously
(multiplexing)
3. The CRISPR/Cas9 system enables efficiency and high
specificity with minimal off-target effects of unwanted chromosomal
translocations when well-designed gRNAs are used
The Cas9
requires precise homology between the gRNA and the targeted DNA
sequence, but it does allow a few mismatches of base pairs in the target
sequence when a DBS is generated (4). Depending on the number, position
and distribution of mismatches, this could affect specificity and the
desired application (1). These off-target effects and their long-term
consequences are the current concerns with the CRISPR systems.
Scientists are working on various methodologies, such as Cas9 nickase,
to target single-strand breaks on opposite sides of the targeted DNA
(1,4) – or choosing unique target sequences and optimising gRNA and Cas9
to minimise this phenomenon (5).
Discovery of the CRISPR/Cas9
system can be credited to two principal researchers: Jennifer Doudna, an
award-winning scientist from the University of California and the Broad
Institute, Massachusetts; and Emmanuelle Charpentier, a worldleading
expert in the regulatory mechanisms underlying the processes of
infection and immunity in bacterial pathogens – along with each of their
teams. Both scientists have been shortlisted for the Nobel Prize (6).
In
the laboratory, geneticists have been able to cut out HIV, correct
sickle cell anaemia and alter cancer cells to make them more susceptible
to chemotherapy (7). In the future, this technology may also make it
possible to edit any human gene.
New Contender
Recently,
Feng Zhang at the Broad Institute – who also worked on the CRISPR/Cas9
system – published his work in collaboration with various other
institutions on the system using the enzyme Cpf1 instead of Cas9. It is
predicted that this enzyme will help overcome some of the difficulties
encountered by researchers as they develop CRISPR-related treatments for
human diseases (8). Numerous companies have been working with the
CRISPR/ Cas9 technology to create therapeutic treatments; however, none
of these have yet reached clinical trials. While Cpf1 is still in the
early research stage, Zhang believes that “there is little doubt
that…there are additional systems with distinctive characteristics that
await exploration and could further enhance genome editing and other
areas of biotechnology, as well as shed light on the evolution of these
defense systems.” (8)
Zhang offers the following perspective on why Cpf1 might be more advantageous than Cas9:
●
Cpf1 uses only one strand of RNA compared to Cas9, which uses two
strands to guide and reach its target gene. A single-strand system might
be simpler, in addition to offering a cheaper design and an easier
delivery process (enzyme-guide complex) into the cells (8,9)
● Cpf1
makes staggered double-strand cuts, or ‘sticky ends’, in the target DNA,
whereas Cas9 cuts both DNA strands in the same location (‘blunt ends’)
once it is delivered into the cell’s nucleus. Zhang et al writes that
the staggered ends make it easier to insert a new gene after the old one
is removed, because these unpaired nucleotides in that gene could only
bond in the desired location. This could overcome the hurdles of Cas9,
where scientists have said that replacing an old gene with a new one
using Cas9 has proved more difficult than simply cutting out a gene
(8,9)
● When Cpf1 hones in on a gene, it makes the cut-off to the
side or further down the DNA strand. Zhang and his colleagues report
that this feature could be “potentially useful” in preserving the target
site for subsequent rounds of editing (8,9)
● In the Cpf1 protein,
Zhang et al identified two enzymes from the Cpf1-family proteins:
Acidaminococcus and Lachnospiraceae bacterium with efficient
genome-editing activity in human cells. The Cpf1-family proteins are
diverse in bacterial species, due to the T-rich PAMs of the Cpf1-family.
This allows for applications in genome editing in organisms where all
characterised mammalian genome-editing proteins require the presence of
at least one G, so the T- and T/C-dependent PAMs of Cpf1-family proteins
expand the targeting range of RNA-guided genome editing nucleases – one
of the primary functions of CRISPR systems. For Cas9, only a small
fraction of bacterial nucleases can function efficiently when
heterologously expressed in mammalian cells (9)
● Francisella Cpf1(FnCpf1) and crRNA alone are sufficient for mediating DNA
targeting. Compare this to Cas9, which requires both crRNA and tracrRNA
to mediate targeted DNA interference – thus making this feature a way to
simplify the design and delivery of genome-editing tools (9)
Curing Single Genetic Disorders
It
may be a while before gene editing can be applied to humans, and this
step should be taken with extreme caution. Diseases involving more than
one gene are so complex that implementation may require more progressive
technology. Any mis-step may result in long-term adverse consequences.
If
this can be achieved, medicine will change. Physicians will learn more
efficient methods of diagnosing and treating diseases – and many
now-common conditions could potentially be eliminated. Prevention would
then be defined as editing a gene for a particular disease once it is
diagnosed. Insurance providers will be more likely to pay for
reimbursement of this new and highly specific preventive medicine,
because once a disease is cured, the associated treatment expenses will
fall over both the short term and long term. However, the one area that
science will not be able to change any time in the extended future is
the ageing process.
Weighing up the Pros and Cons
While
advancing scientific technology is essential for curing diseases and
improving crop yields, we must proceed with utmost care. Although
scientists have the best intentions as they work diligently to advance
medical research, there will inevitably be consequences to every action –
both good and bad. Gene editing may allow people to live longer and
with a better quality of life, but would this also create negative
consequences, such as overpopulation? Or will Mother Nature find a way
to maintain a balance with earthquakes, tornadoes and tsunamis – or with
human-made disasters, such as terrorism and war?
Meanwhile,
consumer technology entrepreneurs have simultaneously introduced devices
such as radar guns that are employed in vehicle traffic control,
microwave ovens and mobile phones – and are increasingly being used even
in less affluent societies. While there are clear benefits to all these
innovations, they also come with a downside, according to studies that
associate their long-term use or exposure with the risk of cancer.
Medical advances may offer the potential for a longer life, but both
humans and Mother Nature have the ability to reverse these benefits.
Sometimes
we become so enamoured with technology that the possibility of its
unforeseen consequences to society are all but forgotten. Can the
repercussions be anticipated, and can defensive measures be put in place
to help avoid these? What has been learnt from our experience with
genetically modified foods and high-tech devices that are supposed to
make life easier? Future generations will likewise be impacted by
consequences rooted in today’s technology. Life itself is a cascade or a
domino effect; everything is related to and affected by everything
else, much like systems biology.
References
1. Hsu P, Landers E and Zhang F, Leading edge review: Development and applications of CRISPR-Cas9 for genome engineering, Cell 157(6): pp1,262-1,278, June 2014. Visit: http://dx.doi.org/10.1016/j.cell.2014.05.010
2. Harrison M, Jenkins B and O'Connor-Giles KM, Review: A CRISPR view of development, Genes and Development 28: pp1,859-1,872, 2014. Visit: http://genesdev.cshlp.org/content/28/17/1859.full.pdf
3. Gaj T, Gersbach C and Barbas III C, ZFN, TALEN and CRISPR/Cas-based methods for genome engineering, Cell 31(7): pp397- 405, July 2013. Visit: www.cell.com/trends/biotechnology/pdf/S0167-7799%2813%2900087-5.pdf
4. CRISPR in the lab: A practical guide. Visit: www.addgene.org/CRISPR/guide
5. Cho SW, Kim S and Kim Y, Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases, Genome Res 24(1): pp132-141, January 2014. Visit: www.ncbi.nlm.nih.gov/pmc/articles/pmc3875854
6. Steenhusen J, Nobel Prize predictions see honors for gene editing technology, Reuters,
September 2015. Visit:
www.reuters.com/article/2015/09/24/us-nobel-predictions-thomsonreuters-iduskcn0ro0bb20150924#jcp4msqk2f3xitcu.99
7. King MC, Charpentier E and Doudna J, April 2015. Visit:
www.time.com/3822554/emmanuelle-charpentier-jennifer-doudna-2015-time-100
8. Lash A, CRISPR update could make gene edits easier, discoverers
say, September 2015. Visit:
www.xconomy.com/boston/2015/09/25/crispr-update-could-make-gene-edits-easierdiscoverers-say
9. Zetsche B et al, Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system, Cell 4:163(3): pp759-771, October 2015 |
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