Biological and Clinical Sciences Research
Journal
Biol. Clin. Sci. Res. J. Volume, 2020:
e017
THE JOURNEY OF CRISPR-CAS9 FROM
BACTERIAL DEFENSE MECHANISM TO A GENE EDITING TOOL IN BOTH ANIMALS AND PLANTS
TAHIR T, *ALI Q, RASHID MS, MALIK A
Institute of
Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan
Corresponding
author emails: saim1692@gmail.com
Abstract
Today
we can use multiple of endonucleases for genome editing which has become very
important and used in number of applications. We use sequence specific
molecular scissors out of which, most important are mega nucleases, zinc finger
nucleases, TALENS (Transcription Activator Like-Effector Nucleases) and
CRISPR-Cas9 which is currently the most famous due to a number of reasons, they
are cheap, easy to build, very specific in nature and their success rate in
plants and animals is also high. Who knew that one day these CRISPR discovered
as a part of immune system of bacteria will be this much worthwhile in the
field of genetic engineering? This review interprets the science behind their
mechanism and how several advancements were made with the passage of time to
make them more efficient for the assigned job.
Keywords: CRISPR-Cas9,
endonucleases, gene editing, immune system, zinc finger, TALENS
Introduction
It
is foreseen that the world’s human population will increase to 10 billion by
2050 and with the increasing population the global requirement of food will
grow to 25% to 70% of the current production rate (Hunter et al., 2017). In order to feed the world it is important to fight
against pathogens which are resulting in yield loss of 20-40% around the globe (Savary
et al., 2012). To overcome the
challenges including increase in yield, pathogen resistance, crop improvement
it was important to develop some techniques that could replace the conventional
selective breeding and induce changes in the genetic makeup of crops. Up till
1970s it was not possible to achieve successful genetically modified organisms.
The first transgenic organism that came into existence was by inserting an
exogenous DNA into the plasmid of E. coli
without interrupting any of bacteria’s own biological function (Cohen et al., 1973). However this was risky
process as there were possibilities that the exogenous DNA may interfere with
the bacterial DNA and mutate it. There were also the chances of mismatches. Then
a technique came, in which the DNA sequence to be delivered were homologous to
the sequence that was targeted in the other organism thus giving more
specificity and less chances of mismatches. This particular technique was the
footstep of gene editing technology (Szostak et al., 1983). Its widespread use was limited due to its inefficiency
but it was used widely for research purposes. Under the following decades after
collecting so much knowledge and after so many studies came the genome editing
through programmable endonucleases, which is the most advanced and recent
technique. These endonucleases for plants include zinc finger nucleases,
transcription effector like nucleases, and CRISPR-Cas9 (Shah et al., 2018; Bao et al., 2019). CRISPR is a ground breaking tool due to its
potential to treat human diseases and edit human genome. It has helped to
identify and understand the procedure of diseases (Zhou et al., 2014), It has helped generate animal disease models (Xue et al., 2014). It has assisted in
advancing genetic engineering in crops (Zhang et al. 2017). The objective of this mini review is to highlight the
features of CRISPR, its advantages and drawbacks over other techniques and to
know the reason behind all the fame and publicity and also some of the work
done on specific crops.
History and
origin of CRISPR
Recently,
CRISPR-Cas9 are stealing the show due to their number of attributes. Ishino was
the scientist who was analyzing the gene that would conduct isozyme conversion
of alkaline phosphatase about 30 years ago and while performing his experiment, he had no idea that he
would discover the first ever CRISPRs
from Escherichia coli (Ishino et al.,
1987). By that time it was very mysterious and unexpected, scientist couldn’t
find out the function of these repeated sequences because the human genome
project did not begin its function of sequencing the genome of organisms that
have therapeutic importance at that time. Then after being detected in bacteria
they were for the first time seen in archaea in 1993, more precisely in
Haloferax mediterranei and the man behind the discovery was Mojica who also
found out after some years that the function of these sequences found in
bacteria and archaea is similar (Mojica et
al., 1993;2000) Janses named them CRISPR in 2002 (Janses et al., 2002) Then four years later
Eugene conveyed that the CRISPR-Cas9 system was a prokaryotic RNA
interference–based immune system (Makarova et
al., 2006). In 2007 it was proven practically that they are truly part of
the adaptive defensive mechanism of prokaryotes while using the lactic acid
bacterium Streptococcus thermophiles (Barrangou et al., 2007). In short in 2012 the face of CRISPR was unveiled and
by this time scientist knew how they could use this system for cutting the target
DNA outside the cell (Gasiunas et al.,
2012). Then the very next year this system taken from streptococcus pyogenes was applied to genome edit human nerve and
the kidney cells of mouse (Cong, Mali et
al., 2013). Beginning an era of targeted genome editing which is easier in
handling.
Structure of CRISPR
loci
Clustered
regularly interspaced short palindromic repeats are found in bacteria and
archea as the part of their immune system, they were first analyzed in Streptococcus pyogenes and classified as
type 2 (Langner et al., 2018).
Streptococcus pyognenes is known to cause lethal infections in humans from less
severe sore throat to a range of infections. It belongs to the gram positive
type of bacteria and works by making colonies in the pharynx (part of throat)
and skin (Rosinski-Chupin et al.,
2019). These are a family of DNA sequences consisting of 2 important
components, Cas 9 endonuclease and single guide RNA (gRNA) which further
consist of pre-crRNA and the trans-activating crRNA also known as tracr-RNA
(Weeks et al., 2016). Cas 9 endonuclease
is a protein and as the name indicates it is an enzyme involved in cleavage,
the customizable guide RNA is to escort the enzyme to the site of target sequence (Jiang and Doudna, 2017; Khatodia
et al., 2016). As the name clearly
shows CRISPR consist of short repeats that are 28-37 bp sequences in length (Barrangou
et al., 2014), these sequences are
unique from each other but are of similar length and are separated from each
other from spacers. Every repeat is arranged in a palindromic manner. These
Spacers play a major role in bacterial immunity as they store the memory of the
sequences from all the past attacks. The number of these spacer vary from specie
to specie it can be from 1 to 100 (Grissa et
al.2007). The protospacer adjacent motif (PAM) for streptococcus pyogenes
is NGG .The PAM are present downstream of target DNA (Jiang and Doudna, 2017).
Further the Cas 9 enzyme consists of two nuclease domain which induces a double
stranded break as one domain binds to the target DNA strand which is homologous
to the guide RNA and the other domain binds with the non-target strand (Gao et al., 2017).
Remodeling CRISPR
for genetic engineering
The
double stranded break efficiently induced as a result of cleavage activity of
Cas 9 enzyme is the main idea behind the diversity in genetic outcomes using
these technologies. After the cleavage cellular repair mechanism is activated
that is of two types HDR and NHEJ. In case of non-homologous end joining (NHEJ)
random DNA pieces are arranged at the end of double stranded break and then are
joined by cell’s own repair machinery. This method is active in all cycles of
cell and is more prone to errors (Moore and Habor 1996). This process does not
require homology templates as they directly ligate break ends. They can further
cause indels (insertion or deletions) in the region of DSB, this can be used to
achieve frameshift mutations (Waters et
al., 2014). However NHEJ is quite unspecific and random process and is not
efficient for single base gene knockout or insertion. Alternatively Homology
directed repair occurs when sister chromatids are present. This DNA template
contains the Cas 9 and gRNA along with the DNA sequence to be delivered into
the cell. This method is more specific and less prone to errors (Pardo et al., 2009). However these site
specific enzymes are very versatile in nature as in addition to gene editing
they are also used as an artificial transcriptional factors. Scientist
introduced two mutations in the cleavage domains of cas 9 nuclease which
confiscated its cleavage activity but it was still able to bind to the DNA. In
this way these systems were used to increase or decrease a gene expression by controlling
its transcription (Bikard et al.,
2013).
Brief discussion
of other nuclease
Mega nucleases
Starting
with the mega nucleases which are also called homing endonucleases, are site
specific like other restriction endonucleases. They have the ability to
recognize large target DNA sequence that can be greater than 12 bp (Stoddard
2005). Discovered in 1990 , they have proved themselves to be an efficient tool
for genome editing as they can generate
homologous recombination (Epinat et
al., 2003) and desired alterations (Arnould et al., 2007). They possess five families out of which most
important one is LAGLIDADG (Stoddard et
al., 2006). As they can induce homologous recombination, they were used in
a number of organisms including drosophila, E.coli, mice, plants and
trypanosomes (Paques 2007). These mega nucleases are very specific in nature
which gives them prescion and show very less level of off targeting effects,
cell toxicity but changing the target of mega nucleases requires a large amount
of work to obtain the results (Takeuchi et
al., 2014; Thyme et al., 2014;
Smith et al., 2006).
Zinc finger
nucleases
As
we know that zinc finger proteins are widely being used by nature (Bedis et al., 2009), so there must be some
advantages of its this much use in nature that could be manipulated and reused
in the form of artificial enzymes such as zinc finger nucleases. Zinc finger nucleases
have been very successful in the field of genome engineering. These
extraordinary enzymes consist of customizable zinc protein domain and a
cleavage domain of FokI restriction endonuclease (Kim et al., 1996). A single zinc finger contains 28-30 amino acids that
have a remarkable ability to functionally vary and structural flexibility to
bind any triplet depending upon the form DNA sequence (Weirauch et al., 2010). These were the first of
all the exonucleases that were used in a large amount (Porteus and Carroll
2005; Urnov et al., 2010) This genome
editing artificial endonuclease has been successfully used in a number of
organisms including maize (Shukla et al.,
2009) Drosophilla (Beumer et al.,
2008) rat (Geurts et al., 2009) most
importantly ZFN are in clinical phase trial 2 for the treatments of AIDS
proving themselves to be helpful in gene therapy (Cannon et al., 2011). But in spite of all these pros, there are always
some limitations associated with a technology and as for ZFNs it is their off
target effects (Gabriel et al., 2011;
Pattanayak et al., 2011). They are
also difficult to construct as compared to more recent and advanced gene
editing techniques.
TALENS
Next
came the TALENS, these are the proteins found in a bacteria called Xanthomonas that
is toxic to plants as it infects them (Moscou et al., 2009; Botch et al.,
2009). TALENs and ZFNs are a bit common in nature as they share some similar
features, for instance a cleavage domain of FokI is found in both (Kim 2014).
In addition to nuclease domain TALENs consist of a TALE DNA-binding region,
which is a repeating unit having about 34 amino acids. The connection between
the target DNA and the recognition domain is made possible by the repeat
valuable diresidues that are the amino acids present at 12th and 13th
position (Boch et al., 2009; Moscou and Bogdanove 2009).This
exceptional nature and TALEs ability to recognize DNA length of 12-20bps give
them specificity in genome editing (Guilinger et al., 2014). After some research on these molecular scissors, it
came into light that these are more specific in nature and shows less off
target effects (Wang et al., 2015).
Another quality of them making them more acceptable than ZFNs is that there is
no need of directed evolution for them thereby saving a lot of time and
experience of combining an enzyme to make it functional (Mussolino et al., 2014).
Why CRISPR is preferred
over others?
Due
to so many reasons CRISPR has replaced ZFN and TALENS. At number of times
CRISPR systems have proved themselves more advanced and trustworthy over other
techniques (Sander and Joung 2014). For instance ZFNs and TALENs require a
customizable protein which will guide them to the target site. The designing
and engineering of this protein is complex, time taking and expensive. On the
other hand CRISPR depends only on the engineering of short guide RNAs (Sternberg
et al., 2014). Multiplex gene
changing is also very difficult using other two techniques as specific proteins
are needed for each gene but by using CRISPR systems multiple gene editing is
handy as only many guide RNAs are to be delivered in the cell (Campa et al., 2019). Additionally there is
this fact that both ZFNs and TALENs work as a dimer to generate a double
stranded break at the target site , this can be a problem as loading capacity
of some of the vectors is less, so their delivery can be a great hurdle.
However the delivery of CRIPSR systems is more obstacles free (Wu et al., 2010).
First
application of CRISPR in human beings
Previously,
genome editing was labor intensive; it could take months, costly and often
limited to labs only under the supervision of experts. But now with the
emergence of nucleases and advanced gene editing technologies which are
friendly, cost effective and takes only weeks, it has become possible to delete
the genes, (Lee et al., 2010) insert the genes (Moehle et al., 2007), replacing the faulty genes (Ran et al., 2013) and even rearrangement of chromosomes (Torres et al., 2014). Experiments using
CRISPR started occurring on other mammals excluding humans. Because
experimenting on human beings is always a risky thing to do. In early 2018 a
Chinese scientist, Dr. He Jiankui started experimenting to produce babies from
embryos that were genetically edited. These were the babies with modifications
against HIV. HJ named them Lulu and Nana, In this case a specially constructed
CRISPR was injected into the embryo, whose aim was to cause a 32-bp-deletion in
a gene called CCR5.According to Dr. HJ this deletion would cause a nonfunctional
CCR5 protein and it would be impossible to contract with AIDs (Antonio
Regalado:2019). This sounds great but there are a lot of
risks and concerns associated with the procedure as it has been reported that
the efficiency rate using CRISPR is only 15% for the for a single gene
correction (Liang et al., 2015).
One of the major drawbacks using CRISPR editing technique is its off targets
effects and it has been reported by researchers that this can cause
disabilities and unfortunately can even cause cancer in some cases (Kim et al., 2015; de Miguel Beriain and del
Cano, 2018). This means that the doctor himself knew all the possible side
effects of these experiments but did not fully make the parents aware. In short
this technique is not fully safe to be experimented on reproductive cells.
First
application of CRISPR in plants
In august 2012 8 brief reports on the application of CRISPR
system were published (Nekrasov et al.,
2013; Jiang et al., 2013), One of
them included observation on transgenic rice that was intentionally mutated to
increase the product and growth. However the researchers later revealed that
the mutation on the particular gene was successful by using the Cas9/sgRNA
system (Miao et al., 2013; Zhang et al., 2014; Wolt et al., 2016). The plant model species that were used with CRISPR
were Arabidopsis Heynh., Oryza L., and Nicotiana L. (Jiang et al., 2013; Shan et al.,
2013; Xie and Yang, 2013). However CRISPR gene editing in plants have shown all
type of plants problem solved from herbicide resistance to industrial
consumption, from increased yield to crop betterment and at the last increased resistance
to both biotic and abiotic stresses (Modrzejewski et al., 2019).
Limitations of
CRISPR and the ways in which they were improved
As
we know CRISPR is under the spotlight nowadays but there are a number of
factors that influence its activity. And it is important to consider these
factors in order to use this technique for in vivo human gene therapy because
that is a delicate procedure.
Site selection
for targeted DNA
Crispr/cas9
has the ability to recognize and select 23 bp of nucleotides that contains a
PAM sequence on either templates of DNA. In case of SpCas9 this PAM sequence is
said to occur after every 8bp on an average estimation (Ramakrishna et al., 2014). This PAM motif is
specific for every species, as in case of Neisseria meningitides PAM sequence
is appeared to be 5′-NNNNGATT-3′ (Jiang et al., 2013; Ma et al.,
2014). This feature shows a greater flexibility and higher specificity in site
selection which will gradually increase with the discovery of new Cas 9 with
different PAM pattern. However many reports have revealed that this is not as
simple and easy as it appears to be. One report even suggests that CRISPR is
not as specific as ZFNs and TALENS because it can target a shorter sequence.
Hence greater the target sequence greater will be the specificity (Cradick et al., 2013).
Off target
cleavage
Cutting
outside the target sequence is caused by the sgRNA, Cas9 protein has no role in
it but still if we improve the cas9 protein it can reduce these effects. The
journey of improving the Cas9 system has led to mutant Cas9 systems. One of
this system includes the distorting of the cas9 protein in such a manner that
it only produces single stranded breaks. Then the 2 nickase enzymes (CRISPR/cas9)
are used to surround the target site as one binds with the forward DNA sequence
and the other with the reverse DNA sequence. In this way double stranded breaks
are induced with the help of nicks. The single stranded breaks produced by off
target cutting are then fused together simply by the help of DNA ligase. This
mutant system when used in mammalian cells decreased off target cleavage to
three folds with little to no reduction in on target efficiency (Mali et al., 2013; Ran et al., 2013; Cho et al.,
2014). Another mutant system includes the joining of Cas9 with TALEs that have
the ability to recognize and bind to any target site with improved specificity and
reduced off target effects (Bolukbasi et
al., 2015). Additionally, these mutant cas9 proteins are intentionally
designed to weaken the joining of the Cas9 with target DNA strand (Kleinstiver et al., 2016) or the non-target DNA
strand (Slaymaker et al., 2016)
meanwhile keeping the strong on-target cleavage which is the sole purpose to
reduce the unwanted DNA contacts.
Frequency of HDR
After
a double stranded break, there are very less chances of a homology directed
repair in mammalian cells and more chances of nonhomologous end joining, proved
through an experiment in mice where Cas9 based gene editing revealed that the
rate of occurrence of NHEJ associated repair was 20-60% while of HDR was only
0.5-20%, even in the presence of sister chromatids, there are more chances of
NHEJ repair mechanism occurring while using CRISPR system (Maruyama et al., 2015). With the passage of time
many new methods have been adopted to enhance the use of HDR and suppress the
efficiency of NHEJ because it is more prone to errors and for that purpose
small suppressor molecules of NHEJ are used (Yu et al., 2015) gene silencing is being done to prevent the
expression of a particular gene (Chu et
al., 2015) cell cycles are being adjusted or harmonized (Lin et al., 2014). These inhibitors of NHEJ
are very effective as the most common; Scr7 has shown increase in rate of HDR
to up to 19 folds in case of CRISPR based editing (Vartak & Raghavan,
2015). But these suppressors may have a toxic effect on host cells so to
overcome this chemical suppression , alternative ways are being studied like
synchronizing cell into cell cycles (late S phase and G2 phase) where HDR can
occur (Lin et al., 2014).
Action of cas9
Each
cas9 protein from different species have different activity and a specific PAM
sequence. Thus selection of a specific Cas9 is very important as it plays a
great role in efficient gene editing. Such cas9 proteins are identified in a
number of species including Staphylococcus aureus (SaCas9) (Ran et al., 2013) and S. thermophiles (St1Cas9) (Kleinstiver et al., 2015). Cas9 must be transferred to nucleus for the improved
activity and for this a (NLS) nuclear location signal is attached to the Cas9
protein. This has shown improved DNA cutting activity (Shen et al., 2013). Another trick to increase
the cleavage activity is to enhance the single guide RNA (sgRNA) concentration
to Cas9 protein (Kim et al., 2014).
But if increased outside the limit, it may become a cause of off-target cutting
(Fu et al., 2013). The catalytic
activity of Cas9 is low when we compare it to other enzymes (Jinek et al., 2012). But this doesn’t mean
that it is not useful, this feature can be beneficial for a number of things
like gene suppression/activation and short term gene editing with less off
target effects. But not required for other applications where catalytic
activity is the sole purpose.
Some advanced
CRISR systems
Keeping
in view the limitations of CRISPR, scientists developed some enhanced versions
of CRISPR to increase their specificity and efficiency.
CPF1 (Cas12a)
These
class 2 CRISPR systems including Cas9 and Cas12a share a number of common
features that to generate a double stranded break they both rely on RNA
molecules (Fonfara et al., 2016). But
Cas12a recognizes its target site with the help of a single RNA (CrRNA)
molecule in comparison to Cas9 that uses CrRNA:TracrRNA. The cuts produced
using CPF1 have sticky ends while that of Cas9 produces blunt ends with no
overhangs (Zetsche et al., 2015).Lastly
Cas9 has the ability to recognize PAM sequences that have more guanosine ,on
the other hand Cas12a recognizes PAM sequence rich in thymine (Gao et al.2017). After some reports it was
declared that Cas12a show less off target effects and are more specific in
nature which is a major plus point in field of genome engineering (Kim et al.2016) Cpf1 has been successfully
used for genome editing in plants rice and tobacco in the year 2016 (Endo et al., 2016). The simplicity and
specificity of these tools make them very important to be used in base
manipulation, multiplex gene targeting (Bayat et al., 2018) and also this has been used in clinical trials which
is a major achievement (Li et al., 2020).
Cas9n
As
described earlier there are some limitations of CRISPR-Cas9 system just like it
does not have important NHEJ components , therefore when used in some bacteria
it may cause death after cleavage. In such cases we use a mutant of Cas9 called
Cas9 nikase (Xu et al., 2015). Cas9
has two sites for cleavage but if we replace one of the sites with a single amino
acid it produces Cas9n which is capable of inducing a single stranded cut on
the target site (Cong et al., 2013). These
mutant tools have proved themselves in the long run due to a number of reasons,
the first one being their high efficiency, they are easy to construct, are
pocket friendly and does not require a lot of time in construction (Jiang et al., 2013;Ran et al., 2013). Lastly they are being used to reduce the off target
effects by using a double nicking technique (Shen et al., 2014).
Cas13a (C2c2)
Cas13a
is another achievement in the path of CRISPR editing technology. It belongs to
CRISPR class 2 system and its cleavage activity depends upon two domains known
as HEPN (Abudayyeh et al., 2016). The
common feature of Cas13a and Cas12a is the ability to target and edit multiple
loci due to its own single crRNA template (Abudayyeh et al., 2017). This tool can be utilized to control post
transcriptional suppression (Elbashir et
al., 2001). When compared with RNA interference processes Cas13a is more
efficient and more specific (Abudayyeh et
al., 2017). As a result of alternative splicing DNA is split into many
isoforms which are affected when targeted with CRISPR, but with Cas13a it is
possible to target and study the task of single isoforms without affecting
others (Mahas et al., 2018). Similarly
there are so many other novel and enhanced systems of CRISPR to overcome the
drawbacks.
Delivery of
CRISPR into the cells
This
step for successful genome editing is very important as the cells to be altered
must receive these CRISPR systems in order to get modified. For this purpose
there are a number of methods out of which some are more successful for
research work and some are more beneficial for therapeutic and clinical use.
Microinjection
This
is a physical method of delivery having efficiency up to 100% (Horii et al., 2014). It requires a microscope
and 0.5–5.0 μm diameter glass needle which pierce the cell membrane
and the material is delivered directly into the target site of the cell but
while doing so it also damages the cell and it can only infect one cell at one
time which makes it very time consuming and laborious (Zhang et al., 2008). Microinjection is best
suited for in vitro and ex vivo applications.
Electroporation
It
is another traditional method of delivery that uses electrical current that
temporarily generates nanometer pores in the membrane to make it permeable so
that the material could enter the cell. While other delivery techniques depend
upon cell type, this technique is less dependent on it. It is mostly used for
in vitro work. As the procedure
sometimes require high voltage current therefore it is not suitable for in
vivo. Multiple cells can be edited using electroporation at one time (Kaneko et al., 2014). Because of its readily
available sources in lab and high efficiency this technique promises a
continued use for efficient delivery of CRISPR/Cas9.
Hydrodynamics
It is also a physical means of in vivo delivery,
mostly enriched for liver cells (Yin et
al., 2014). It uses a large volume of CRISPR/Cas9 editing system quickly
injected into the blood stream of an animal, This rapid action causes an
increase in hydrodynamic pressure due to which permeability of the cell
membrane is enhanced for the time being and it allows the entry of the desired
material. Mostly tail vein of the mice is preferred to be injected. The simplicity
of this technique makes it a desirable method and it has proved itself a number
of times including in vivo correction of Fah in mouse liver cells (Yin et al.2014).Then later Guan repaired
hemostasis in treated mice, it is a condition in which the blood is stopped to flow
out of the damaged vessel (Guan et al.,
2016). Work on hepatitis B infected mice has also been done using hydrodynamic
delivery method to stop the division and expression of the virus (Zhen et al., 2015). Regardless of these
successful applications of this method , it is still not considered to be used
in clinical works as it can cause some difficulties including stopping of the
heart, enlargement of the liver and increase in blood pressure (Suda et al., 2007;Bonamassa et al., 2011). Transfection rates are
low for this type of delivery and only some type more precisely liver cells are
more capable of successful delivery.
Viral Vectors:
Previous
physical methods discussed cannot be used in human gene therapy because they
are less efficient and they are imperfect (Valsalakumarit et al., 2013). So we have other options of delivery that are:
Adeno-associated
viruses (AAV)
As
viruses can be DNA or RNA based, this one is single stranded DNA based virus
that has been broadly utilized for gene therapy (Daya and Berns, 2008; Samulski
and Muzyczka, 2014).These vectors are being used for multiple gene editing to
study gene function in vivo (Swiech et al.,
2015). It can infect many cells with distinctive specificities. The thing that
makes AAV so special means of delivery is that they are not reported to cause
any disease in human beings and also they are very efficient in their work that
they do not give rise to innate or adaptive immune response or any sort of
linked toxicity (Daya and Berns, 2008). AAV are so flexible that their function
can be seen in in vitro, ex vivo, in vivo. It has a unique feature that the
genomic material delivered through AAV can exist in two forms in the host,
either directly inserted into the DNA after some modifications or outside the
DNA into the cell (Deyle and Russell, 2009). The genomic material that could be
packed inside the AAV particle is only 4.5-5 kb (Wu et al., 2010), which makes the packaging quite challenging but for
now there are so many ways developed to adjust the packaging.
Lenti (LV) and
adeno viruses (AdV)
LV
and ADV are quite different from each other but the main reason they are being
described under the same heading is that their way of delivering Cas9 system is
quite similar. The major advantage of using LV or Adv delivery systems over AAV
is that they have additional packaging space due to their large particle size
that is 80-100nm while of AAV is only 20nm. This feature allows them to adjust
and carry large insertions. These methods of delivery are being used by several
groups such as for gene silencing (Voets et
al., (2017), moreover (Kabadi et al.,
(2014) made a different combination of lentivirus/CRISPR Cas9 that has the
possibility to be used for delivery in invivo. Viral vectors including LV are
used to screen the function of genes more specifically they identified the
genes that are important for cell death induced by West-Nile-Virus (Ma et al., 2015).These viral vectors have
great applications in clinical studies but there are a number of obstacles in
their way like they evoke immunogenicity (Follenzi et al., 2007; Ahi et al.,
2011). Additionally there are chances of off target effects and cell damage
(Bestor, 2000; Papapetrou and Schambach, 2016). So they must be handled with
care when using for genome editing.
Non
viral-vectors
There
are so many non-viral vector delivery methods that are very efficient in
replacing the physical and viral methods some of them are as under.
Lipid
nanoparticles
They
are also known as liposomes. These particles exhibit a major advantage as like
other non-viral methods that they do not contain viral components, therefore
they do not have safety issues or they do not give rise to immunogenicity. They
can be used for ex vivo, in vivo and in vitro experiments giving a wide range
of applications. While delivering CRISPR/Cas9 there are two ways of using
liposomes either as delivering Cas9 and single guide RNA as genetic material
which will just be like microinjection in result (Yin et al., 2016) or as ribonucleoprotien complexes that contains Cas9 and SgRNA which has proved
successful quite a times (Wang et al.,
2016). In spite of all this when the nanoparticle has passed the surface of
cell its translocation to the nucleus is quite a problem because in the passage
it may come across the lysosomal pathway that will cause the degradation of the
lysosome material which is a failure therefore this method of delivery has very
less efficiency. But they can always be used after some improvement.
Gold
nanoparticles
Another
prospect for delivering ex vivo, in vivo and in vitro includes the use of AuNPs
which when joined with Cas9:SgRNA ribonucleoprotiens make a complex that
increases the efficiency rate of delivery up to 30% for causing the needed mutation
(Mout et al., 2017), This method of
delivery was also used in a mice who had Duchenne muscular dystrophy (DMD) in
which the muscles are weakened due to changings in a protein called dystrophin.
The gene responsible for this mutation was corrected to 5.4% after using this technique of delivery and
also there was restoration of drystrophin gene expression (Lee et al., 2017) Now for the importance
these particles are inert and the will not initiate any sort of immune response
and are always a very good alternative to viral vectors.
DNA nanoclew
it
is a distinctive technique for delivering CRISPR system but it special as it is
not viral and only requires repeating DNA and PEI (polymer polyethylenimine),
it came into existence by (Sun et al.,
2014), A DNA nanoclew is just like a ball of yarn which is rolled on its own
and joined together with the help of palindromic sequences. It has only been
applied in in vitro being a new technique. When it was combined with RNP it
showed 36 % efficiency in an experiment performed in 2015 (Sun et al., 2015).
CRISPR component
delivery in plants
CRISPR-Cas9
constructs can be delivered to explants using Agrobacterium mediated gene
transfer in which the vector consisting of Cas 9 protein and guide RNA is
introduced into the Agrobacterium
tumefaciens (Shan et al., 2018).
Another method of delivery that is used is particle bombardment (Liu et al., 2019). Both systems have
advantages and drawbacks but Agrobacterium
infection is the most used one to obtains transgenic crops due to its
simplicity and low copy number integration but it may cause cell death and
browning of the plant, On the other hand particle bombardment technique is
expensive which limits its use but it can infect a greater number of genotypes
with in a specie as compared to agrobacterium
infection (Altpeter et al., 2016).
Furthermore viral vectors have also been used to deliver CRISPR machinery into
transgenic plants (Ali et al., 2015).
Cas9 can be delivered in a plant cell in the format of DNA, mRNA or proteins. Each
one of them has their own positivities and negativities. For instance DNA form
is low cost and once the DNA is integrated it becomes stable. But both on
target and off target rates are high of this method. If we deliver it in the
form of mRNA, there are chances that the mRNA may be degraded in the way and it
is also not stable after integration, but to produce Cas9 protein from mRNA is
a very quick process. At the end ribonucleo-proteins can be injected directly
(Woo et al., 2015). This method of
delivery can give rise to directly the genome editing steps without wasting
time. But defense mechanism may be active in eukaryotic cells after direct
introduction of bacterial proteins through this method secondly the size
endonuclease size is large which may become a hindrance in the pathway of
introduction into the plant cell (Glass et
al., 2018). In agricultural approaches both mRNA and nucleoprotein forms
are suitable as compared to DNA form of delivery because it introduces
transgenes into the plant.
Application in
plants
Use of CRIPSR in
Plants to combat biotic stress
Every
year a great amount of crop yield is lost due to biotic and abiotic stresses.
In order to fulfill our need of food we use engineered plants that have the
ability to fight against bacteria, viruses, fungus, nematodes, insects and
other pathogens that make up the biotic stress of a plant. There is an
estimated average global yield loss of 11 to 30% just due to these pathogenic
plant microbes and because of insect attack on plants (Savary et al., 2019) and an average 10-15% damage
caused by alone viruses (Van et al., 2008).
So since the very beginning scientists have been trying to develop new ways to
introduce resistant crop varieties in the environment and for this purpose they
used some conventional techniques including cross breeding, hybridization,
biological and chemical mutagenesis (Langer et
al., 2018), but there were several problems with these methods such as
chances of undesired or non-targeted modifications and the process of screening
required a lot of time and labour work. Conventional techniques were followed
by genetically modified and genetically engineered crops which were again
problematic. In last 10 years the hard work of scientists has finally been paid
with the introduction of CRISPR
Table 1A list of
some examples of biotic stress pathogen that has been edited using CRISPR
Pathogen |
Disease |
Host |
Target Gene |
References |
Oidium neolycopersici |
powdery mildew fungal |
Tomato |
Deletion in MLO1 locus |
Klimek-Chodacka et al., (2018) |
Pseudomonas syringae pv. |
Bacterial
disease |
Tomato |
SlDMR6-1
gene is edited |
Wang
et al., (2019); Thomazella et al., (2016) |
rice tungro spherical virus (RTSV)] |
Tungro (RNA) |
Rice |
Translation initiation factor 4 gamma
(eIF4G) gene. |
Macovei et al (2018) |
Turnip mosaic virus (TuMV) |
Viral
(RNA) |
Arabidopsis
thaliana |
eIF(iso)4E
locus |
Pyott
et al., (2016) |
Blumeria graminis f. sp. tritici (Bgt) |
Powdery mildew |
Wheat |
MLO gene |
Wang et al., (2014) |
Erysiphe necator |
Powdery
mildew |
Grapes |
MLO7 |
Pessina
et al., (2016) |
Erwinia amylovora |
Bacterial fire blight |
Apples |
DIPM-1, DIPM-2, and DIPM-4 |
Malnoy et al., (2016) |
Pyricularia oryzae |
Blast
disease |
Japonica
rice |
codons
close to OsERF922 |
Wang
et al., (2016) |
CRISPR has shown
exceptional and promising work in developing successful biotic resistant crops
in such a short period which was not possible by old techniques
CRISPR and
abiotic stress in plants
Similarly
this site specific genome editing technique has been successfully used in 20
crop species (Ricroch et al., 2017)
to either increase their yield of to make them resistance to biotic or abiotic
stresses. Abiotic stress is the most serious obstacle in the development and
production of agriculture. Abiotic stress involves a number of genes and mainly
environmental conditions (Bhat et al.,
2016). These environmental factors include drought, salinity, cold and heavy
metals. Drought and salinity are the major contributors in effecting the crop
productivity as they affect 40 and 7% of the global area (Trenberth et al., 2014). These abiotic stresses affect
all the major and essential processes of a plant including photosynthesis,
protein building, stomata passage and the rate at which transpiration occurs (Zhang
X. et al., 2014; Zhu et al., 2016; Elferjani and
Soolanayakanahally, 2018). Here is a list of plants that have been modified
using CRISPR to make them adopt to these stress conditions.
Table 2. List of CRISPR mediated gene
editing of abiotic stress genes in crops
Crop
Abiotic stress targeted gene References
Maize Drought
tolerance ARGOS8 Shi et
al., (2017) |
Corn Drought tolerance ARGOS8 Shi et
al., (2017) |
Arabidopsis enhanced OST2/AHA1 Osakabe et al., (2017) Stomata
responses |
Tomato Drought Tolerance SlMAPK3 Wang
et al., (2017) |
Rice salinity tolerance OsRR22 Zhang et al., (2019) OsNAC041 Bo et al., (2019) |
Wheat Drought tolerance TaDREB3 Kim et
al., (2019) TaDREB2 |
Using CRISPR to improve plant nutrition and yield
After
fighting with biotic and abiotic challenges in plants the next big challenge
was to improve the crop yield to feed the ever increasing population. There is
reported to be an increase in the nutrition value of sorghum and wheat under
the assistance of CRISPR (Li et al.,
2018a; Zhang et al., 2018b). In 2017
a batch of scientists worked together and they were able to mutate the promoter
region of some quantitative genes of tomato under CRISPR that resulted in so
many improved traits including better fruit shape and size that the non-mutated
(Rodríguez-Leal et al., 2017). From
this study we have the idea that plants traits can be enhanced that just need a
little push by utilizing CRISPR.
Table 3. Some
examples of CRISPR edited crops to enhance crop yield and quality
Plant Trait improvement Targeted gene References
Apple non browning Polyphenol oxidase Halterman et al., (2015) Mushrooms (PPO) genes Nishitani et al., (2016) Potatoes Waltz
et al., (2016) |
Rice increased seed size GW2, GW5, TGW6 Xu et
al., (2016) |
wheat increased seed size TaGW2 Wang et
al., (2018) |
Banana
increased quality RAS-PDS1, Zhang et
al., (2018)
RAS-PDS2 |
Rice increased cooking Waxy gene Kaur et
al., (2018) Increased nutrition |
Rice increased amylose SBEIIb Zhang et
al., (2018) Synthesis |
Wheat low gluten family α–gliadin Sun et
al., (2017) |
Soybean high yield FAD2-1B , Sanchex-Leon et al., (2018) Improved level of oleic
acid FAD2-1A |
Maize increased level PPR and RPL Kim et
al., (2017) Of healthy tryptophan
Qi et al., (2016) And lysine |
These are a very
few success stories of using CRISPR and with the seen progress there are so
many chances of more to come.
Industrial
applications of CRISPR
As
we know that biological industries require microorganism for their various
processes. In order to enhance the efficiency of these microorganisms,
modification was done using laborious and time taking methods. To the surprise
of scientists came CRISPR as a gift to solve most of their problems with gene
editing. Here is a little summary of using them to manipulate important
industrial bacterial, yeast and filamentous fungi cells.
In Bacteria
E.
coli is one of the well-known and significant strains used to produce many
chemicals drugs and other useful biofuels in the industry. With the passage of
time there is a great advancement in every field which has enabled us to
increase the yield and revenue of the desired products from E. coli (Dellomonaco et al., 2010). For instance CRISPR was
availed to uplift the flavonoid production (flavonoids are strong antioxidant
with qualities of anti-inflammation and immunity). It was done so by finely
adjusting and refining the metabolic systems of cell like TCA cycle and
glycolysis. It was proved by this experiment that CRISPR can be used in this
way without interfering with the cell’s normal growth (Wu et al., 2015). In another example for the production of n-butanol,
the expression of four of the cell’s own gene (pta, frdA, ldhA, and adhE) was
reduced using guided CRISPRi system. N-butanol is needed for the synthesis of
ethanol, acetate, and succinate. These are important biofuels (Kim et al., 2017). Other species of bacteria
were also facilitated by CRISPR for example solventogenic Clostridium. These
strains were not efficiently engineered due to some lacking like less
understanding of its biological pathways and structure , secondly transformation
rate was also low (Pyne et al., 2014;
Bruder et al., 2016). During 2017
CRISPR engineering was done in a strain of Clostridium for Increased level of
butanol. The butanol level reached to 19.0 g/L which was the highest record
ever occurred through batch fermentation (Wang et al., 2017). Furthermore our little star was also able to produce
succinate (chemical) in huge amount in Synechococcus
elongates (Li et al., 2016).
In yeast and
fungi
Similarly
yeasts are also widely used for the production of great number beneficial
products like enzymes, artificial food flavoring agents, chemicals,
inexhaustible biofuels and in the production of biopharmaceuticals (Raschmanova
et al., 2018). Yeasts have a tough body
which enables them to even live in unsurvivable environments. For the
production of biopharmaceuticals eukaryotic post translational modifications
are very important but bacteria lacks such modifications, so yeast can be used
instead (Thomas et al., 2013). CRISPR
was applied in many yeast species including Schizos
accharomyces pombe. The purpose of this modification was to achieve more
efficient promoters. Jacob et al was
successful in achieving a high efficiency of 98% in this process and resulted in
highly efficient knockout (Jacobs et al.,
2014). Once again fungi is used to make many pigments, organic acids
polyunsaturated fatty acids and so many other things (Dufossé et al., 2014; Ji et al., 2014) But there were some problems with its usage in
industry Firstly the passage of tool across the fungal cell wall was a big
challenge and even if the gene editing tool was delivered, editing efficiency
was low and thirdly it was a laborious work that required a lot of time.
However it was all until the discovery of CRISPR. CRISPR has been implemented
successfully in Trichoderma reesei (Hao and Su, 2019) and in Aspergillus
fumigatus which achieved a high efficiency in gene editing of 100% (Zhang et al., 2016). This once again showed
the greatness of CRISPR as a power full genome editing tool.
Oncology and
CRISPR
There
are number of genetic and epigenetic factors involved in the cause of malignant
cell formation. These factors can be tumor suppressers, cancer causing genes,
chemo resistant genes and control loci. CRISPR appears to be helpful and
promising in this matter because it possess the ability to correct such
mutations and cure cancer (White and Khalili et al., 2016). Scientist are successful by using targeted CRISPR systems
to generate cell lines and animal models for different type of cancers by
either deleting or inserting the oncogenes or tumor suppresser genes (Platt et al., 2014). These animal models are
also used to study the effect of specific genes in a disease (Chen et al., 2016). The most widely used
animal model is the mice because of its exceptional qualities over other. It takes
lesser time to generate mutants it is cheap to use and it is applicable in large
scale mutagenesis studies (Hsu et al.,
2014). Moreover scientists have been fruitful to generate cell lines for lung
cancer (Choi and Meyerson et al. 2014),
liver cancer (Xue et al., 2014) and
pancreatic cancer (Chiou et al.,
2015). CRISPR can target the oncogenes like receptor tyrosine kinase Erb2 involved
in causing tumor cells (Brown and cooper et
al., 1996). According to recent researches cas9 was employed to knockdown
or repress MDR1 in osteosarcoma cells. As a result there was reduction in
resistance to chemotherapeutic agents which is a good sign (Liu et al., 2016). In another experiment
performed in vivo in mice, NANOG and NANOGP8 genes were targeted by CRISPR.
These genes are take part in causing prostate cancer. However the end result
was reduced tumorigenic concentration (Kawamura et al., 2015).
Terminating
viral cells
This
feature show the worth of CRISPR more as it can be implemented to destroy the
replication system of virus, thus fighting with the viral diseases. It has been
used to target Hepatitis B virus (HBV) (Zeisel et al., 2014). Researchers have been fortunate in targeting with Cas
9 both inside and outside the living organism and producing long term reduction
in viral capacity and less production of antigen that is disease causing (Kennedy
et al., 2015). Through some other
tests researchers were able to knockout part of (HIV-1) from human CD4+ T-cells
(Kaminski et al., 2016). However most
of the cases are in clinical trials.
Fighting with
Genetic diseases
Apart
from combating with cancer, viral and bacterial infections, CRISPR are used to
eliminate and target such inherited genes that causes disorders. For instance
in an eye disease named as Retinitis pigmentosa in which the individual losses
his sight due to the disruption of photoreceptors is successfully being
targeted and mutated with RPGR gene. This gene in healthy individuals expresses
protiens that are involved in vision building factors. It is a hope for all the
blinded patients of Retinitis (Bassuk et
al., 2016). There is a great room for CRISPR editing in other diseases
because of its stable qualities. Other disease like cystic fibrosis that occurs
a result of mutation in CFTR gene (Kerem et
al., 1989) or sickle cell are long lasting disease which shorten the life
span of the one carrying the genes (Lanzkron et al., 2013). With the help of CRISPR scientist were able to edit intestinal stem
cells from the patient of cystic fibrosis outside its own living cell and
repair their function (Schwank et al,.
2013). In case of sickle cell with HDR mediated Cas9 Li and his coworkers have edited
the disease causing genes in pluripotent stem cells as a result no noticeable
off targeting was seen (Li et al,.
2016). Further clinical trials are under process for sickle cell.
Conflict of
interest
The
authors declared absence of any type of conflict of interest for manuscript
publication.
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