GENOME EDITING TECHNOLOGIES: CRISPR, LEAPER, RESTORE, ARCUT, SATI, AND RESCUE
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EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
Review article:
GENOME EDITING TECHNOLOGIES:
CRISPR, LEAPER, RESTORE, ARCUT, SATI, AND RESCUE
Şenay Görücü Yılmaz
Department of Nutrition and Dietetics, Gaziantep University, Gaziantep, Turkey 27310,
E-mail: gorucu@gantep.edu.tr; senaygor@yahoo.com
http://dx.doi.org/10.17179/excli2020-3070
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
Genome editing technologies include techniques used for desired genetic modifications and allow the insertion,
modification or deletion of specific DNA fragments. Recent advances in genome biology offer unprecedented
promise for interdisciplinary collaboration and applications in gene editing. New genome editing technologies
enable specific and efficient genome modifications. The sources that inspire these modifications and already exist
in the genome are DNA degradation enzymes and DNA repair pathways. Six of these recent technologies are the
clustered regularly interspaced short palindromic repeats (CRISPR), leveraging endogenous ADAR for program-
mable editing of RNA (LEAPER), recruiting endogenous ADAR to specific transcripts for oligonucleotide-medi-
ated RNA editing (RESTORE), chemistry-based artificial restriction DNA cutter (ARCUT), single homology arm
donor mediated intron-targeting integration (SATI), RNA editing for specific C-to-U exchange (RESCUE). These
technologies are widely used from various biomedical researches to clinics, agriculture, and allow you to rearrange
genomic sequences, create cell lines and animal models to solve human diseases. This review emphasizes the
characteristics, superiority, limitations, also whether each technology can be used in different biological systems
and the potential application of these systems in the treatment of several human diseases.
Keywords: Genome editing, bioengineering, genome editing technologies, gene therapy
INTRODUCTION specific genetic disease by adding a func-
tional gene to replace the defective gene. To
Genome editing is a genetic engineering
understand the advances in gene-editing tech-
initiative by inserting, deleting, modifying, or
replacing DNA into the genome of a living or- niques, it is necessary briefly to comment on
ganism. In previous technologies, the genetic the techniques before CRISPR. Recognizers
material was randomly inserted into the host and degrading enzymes must be found to en-
genome. Since this randomness has the poten- sure target specificity. Restriction enzymes
tial to disrupt or alter other genes in the organ- used in the process of specific target recogni-
ism, new solutions have been sought. Nowa- tion are called "molecular scissors." These
days, the insertion process can be carried out molecular scissors cleave the DNA into spe-
specifically to the target. Through specifically cific recognition sequences known as cleav-
targeted techniques, off-target effects are re- age sites or nearby regions. The use of endo-
duced and also allow for the regulation of cer- nucleases and exonucleases is common for
cleavage regulation. These nucleases have
tain sequences within a known genome. In an
organism, it has been made possible to cure a two subcategories as deoxyribonucleases and
ribonucleases. Specifically produced nucle-
19EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
ases can be programmed according to the pur- editing focus on the correction of single-gene
pose. Nucleases form double-strand breaks diseases, immunotherapies, cancer, viral in-
(DSBs) in the genome of interest and are then fections and cardiovascular diseases. While
repaired by cellular mechanisms. These repair some of these techniques are applied directly
mechanisms that give the techniques their to the patient, some of them are still in the
name are failure-prone nonhomologous end- clinical trial phase. Here, we review six ge-
joining (NHEJ) and error-free homology di- nome editing technologies and discuss appli-
rected repair (HDR). Thus, they can cause the cations that can be done using a variety of or-
addition, deletion or alteration of nucleo- ganisms and gene editing tools.
tide/nucleotides in the target sequence
(Helena et al., 2018). Mutations caused by re-
MECHANISMS OF GENOME EDITING
pair mechanisms can disrupt, remove or re-
TOOLS
pair the defective region in genes. So what is
our main purpose in using these techniques? CRISPR-CAS SYSTEM: naturally
The process that starts with the treatment of occurring genome editing system
diseases in genome technologies continues CRISPR is a breakthrough that has caught
until the breeding and productivity of plants the attention of almost every scientist and im-
(Friedrichs et al., 2019) and animals (Zhao et agined what could be done. While this tech-
al., 2019). Researchers are developing new nology looks complex, its basis is clear, and it
tools involving gene/genome editing to pre- is effective in correcting genes. In other
vent and treat diseases in humans. Genome words, we can say that CRISPR technology is
editing tools have the potential to treat dis- based on searching for a specific piece of
eases such as cystic fibrosis (Hodges and DNA in a cell and changing this piece of DNA
Conlon, 2019), DMD (Fernandez-Ruiz, (Rath et al., 2015) and can turn genes on or
2020), and diabetes (Balboa et al., 2019). The off without changing the sequences in its tar-
main goal in genome editing is to make it pos- get (Mrowka et al., 2018). CRISPR technol-
sible to treat genetic-based diseases. There- ogy can enable us to treat or prevent many dis-
fore, choosing the appropriate treatment de- eases. When we go to a step further, we can
pends on genome disorder to be treated. The reprogram genomes within the framework of
best way to correct the genome depends on ethical rules against congenital or acquired
the target and/or the type of damage and the diseases with this technology in the ethical
size of the target. The unknown target and framework. The biggest support for technol-
what kind of correction will be encountered ogy is the Cas (Cellular apoptosis susceptibil-
after genetic modifications may be disap- ity) enzyme, found in bacteria and a CRISPR
pointing at the end of the process. Target mol- partner used in defense against viruses (White
ecules in the application of these techniques et al., 2015). Cas9 can guide access to specific
can be DNA, RNA, mitochondrial genome or locations in the genome by searching for a
protein. In addition to these, the target tissue short RNA (Hsu et al., 2014) and can be pro-
or biological resources can be germline, so- grammed to bind to CRISPR (Wilkinson et
matic cell, stem cells or embryos from differ- al., 2019). Using the CRISPR-Cas system,
ent lineages. Different techniques can be used DNA sequences and proteins in the endoge-
alone or in combination, and understanding nous genome can be more easily edited or
the potential of these techniques is important modulated in any selected organism. In addi-
for the effectiveness of genome editing tech- tion to being simple and measurable, the ge-
nologies. The programming of the genome netic repair mediated by Cas-9 also proves the
has greatly accelerated the gene-editing pro- reason for the links between genetic varia-
cess in many areas and uniquely enables re- tions and biological phenotypes. The im-
searchers to perform the maneuver they want portance of CRISPR, found during the exam-
on the genome. Preclinical studies on genome ination of the bacterial genome, comes from
20EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
its matching with some virus sequences. is prevented from slipping on the DNA chain
When bacteria become infected with the vi- and the production of the protein for the target
rus, they store the DNA fragments of the virus gene is prevented. In a sense, the gene is si-
in the CRISPR sequence and thus form a bac- lenced or inactivated. As a result, genes can
terial index (Karginov and Hannon, 2010). In be activated or vice versa completely silenced
this way, they develop an adaptive immune with the CRISPR-Cas system. There are
system. By these mechanisms, bacteria can- many reviews and researches on how the
not get rid of viral infection with this process CRISPR-Cas system works and its history.
alone. Also, the bacterial virus must lyse. At The subject of interest is the contribution of
this point, RNAs synthesized from CRISPR CRISPR technologies to the solution process
sequences (CRISPR RNA-crRNA) and Cas of human diseases and their applications in
proteins are required. crRNAs provide syn- other biological systems.
thesis using sequences that help identify the
virus, and when the virus enters the cell, it CRISPR APPLICATIONS: technical
performs the recognition process by matching approaches with key human diseases
the target sequence with single-stranded The first major steps towards finding so-
RNAs (Hille and Charpentier, 2016). Then lutions to human diseases with CRISPR were
the main mission is to destroy the virus, and taken by two research groups. Using this sys-
this task is performed with the CRISPR-Cas tem, the researchers conducted an in vitro
system. As an RNA-guided protein, Cas's study on mice. In this model, the cause of
goal is to break down double-strand DNA and DMD was the hotspot mutation in the dystro-
bind to the virus genome with CRISPR in the phin gene and the dystrophin reading frame in
cell (Jiang and Doudna, 2017). As a result, the 45-55 exons of the gene was restored us-
Cas cuts the binding sites in its target and de- ing this system (Long et al., 2014; Ousterout
activates the virus (Hryhorowicz et al., 2017). et al., 2015). At the end of the study, DMD
Meanwhile, the bacterium makes copies of mutations were corrected by 62 %. Thus, tar-
the virus genome to add to own account geting the responsible gene in genetic dis-
(Gebre et al., 2018). It synthesizes RNA and eases caused by a single gene and mutation,
protein using the sequence it has copied. In correcting the mutation in the disease and al-
this way, it develops an adaptive immune sys- leviating the clinical manifestations of the dis-
tem mechanism that targets and destroys vi- ease seems to be easier compared with multi-
ruses that invade the cell. This is important factorial diseases. The mutations of genes
because this mechanism in bacteria can be whose genetic basis is changed but, which
used to mediate the human genome. The Cas constitute the center of the disease and are
protein does not bind randomly to target considered to affect the disease, should be
DNAs. It can bind completely to the desired corrected by this mechanism and the results
target through crRNAs and mRNAs. Accord- should be evaluated. The method is advanta-
ing to the technology applied today, the most geous over other systems. Because it is easy
widely used Cas protein is Cas9. For Cas9 to to work, results are obtained in a very short
be functional, the desired sequence must be time, and it is made using the organism's
added and at the same time prevented from mechanisms. The goal today is to correct mul-
truncating the target DNA. After that, the nor- tiple genes and mutations simultaneously or
mal protein synthesis mechanism works and at different times.
proteins are produced from DNA in the pres- The CRISPR-Cas system is an important
ence of polymerase. The polymerase slides therapeutic target used to understand the
along the DNA chain and scans the entire mechanism of known genetic diseases, create
DNA. Thus, protein synthesis takes place. cell and animal models, and mimic diseases.
Cas9 is placed in the desired site on the DNA Genome editing based therapy can provide
with the crRNA. In this way, the polymerase
21EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
restoration of gene function or repair of muta- CRISPR system, the healthy gene was re-
tion. The most easily applicable genome edit- jected by human embryos. This result showed
ing source is SNPs. There are several ap- the researchers that this repair was not a solu-
proaches with different strategies for SNP re- tion, and they suggested that homology repair
pair. Considering the number of bases in the is required to treat cardiomyopathy and some
DNA and the Watson-Crick match, we can re- other inherited genetic diseases (Ma et al.,
place the A, T, C, G nucleotides with 12 pos- 2017). Avoiding indel mutations is extremely
sibilities. Besides, we can also delete and in- important in gene therapy applications, as
sert more than one nucleotide as in the dele- these genomic or chromosomal imbalances
tion of the 4 nucleotides in the HEXA gene can lead to cancer (Rayner et al., 2019; Zhan
that caused Tay-Sachs disease (Min et al., et al., 2019) and other genomic damage
2019) or exons in DMD (Gadalla et al., 2015; (Ghosh et al., 2019). Can this problem be
Tremblay et al., 2016). What is the success overcome in CRISPR applications used in
rate in repairing SNPs when such a process is cancer therapy? Looking at the live system,
implemented? It largely depends on choosing we see that CRISPR is used to treat two can-
the right method. Choosing the right method cer patients with multiple myeloma and sar-
allows for avoiding the editing of unwanted coma (https://www.npr.org/sections/health-
points in the genome. The precision of this ar- shots/2019/04/16/712402435/ First U.S. Pa-
rangement may only be possible if a single tients Treated With CRISPR As Human
cell is involved. However, additional applica- Gene-Editing Trials Get Underway). Scien-
tions are needed for the specificity of the tists know that there should be a limit to this
method. The CRISPR-Cas system has ad- kind of promising work. However, it is also
vantages such as high targeted mutation rate, possible to see news whose ethical dimen-
less cost, simplicity, and high multiplex loca- sions are discussed and the embryo is inter-
tion editing compared to traditional genome fered with (Cyranoski, 2020). It is still diffi-
editing tools including transcription activator- cult to insert target DNA into the CRISPR-
like effector nucleases (TALENs) and zinc- Cas system used in the treatment of embryo
finger nucleases (ZFNs). This system also has intervention, cancer, DMD or similar dis-
negative effects such as creating unwanted eases. Today, one of the most effective solu-
mutations in non-target areas. We can classify tions is the transposons. The CRISPR-associ-
these effects in the CRISPR-Cas system as ated transposase found in Cyanobacteria
targeted (targeting efficiency) and non-tar- Scytonema hofmanni (ShCAST) catalyzes
geted (undesirable). RNA guided DNA transposition. Its effective-
One of the additional applications to be ness is close to 80 % (Strecker et al., 2019).
made to eliminate these effects in the Another important issue in the CRISPR sys-
CRISPR-Cas9 system is the use of single- tem is whether the target is single or double-
guide RNA (sgRNA)/ligand-dependent ribo- strand DNA. This technique has been used for
zymes called aptazime used in plants single-strand DNA.
(Kundert et al., 2019). Unwanted mutations in This doesn't mean we can't use it for
non-human organisms can be prevented with dsDNA. By targeting both dsDNA (DiNapoli
this method. It can also be used as an alterna- et al., 2020) and ssDNA (Bai et al., 2020b)
tive method to significantly reduce the fre- templates in cells, experimental animals
quency of non-target mutations. When the (Pineault et al., 2019), plants (Mao et al.,
CRISPR-Cas system is applied, the most 2019), model organisms (Bai et al., 2020a)
common result is insertion/deletions (Indel and even humans (Ma et al., 2015; Moon et
mutations). For example, in the study where a al., 2019), we can regulate insertions, dele-
healthy gene was added to a gene responsible tions, replication, migration, transformation,
for hypertrophic cardiomyopathy via the copy number loss or gain (Anzalone et al.,
2019) with the CRISPR system. Different Cas
22EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
enzymes are preferred according to the mech- Cas1, Cas2 (Koonin et al., 2017; Makarova
anisms by which they are effective in all these and Koonin, 2015). The biggest problem in
biological structures. the literature is the classification of Cas pro-
tein types. Cas1 (is a metal-dependent DNA-
CAS PROTEINS: Essential for compara- specific endonuclease and produces dsDNA
tive genomic and functional characteriza- fragments (Wiedenheft et al., 2009). Cas 1 is
tion of the adaptive immune system part of the Cas-Cas2 complex, and Cas2 is a
Gene’s editing occurs via mechanisms in dsDNase (Nam et al., 2012) and essential for
which different Cas types are involved. There spacer acquisition in the CRISPR system
are about 45 natural Cas protein families di- (Nuñez et al., 2014). The importance of Cas1
vided into eight subtypes, and each family was understood by increasing sensitivity to
contains 20 proteins. It is divided into three DNA damage and impaired chromosomal
basic classes as I, II, and III (Table 1). These segregation as a result of gene deletion in E.
classes are also named alphabetically within coli (Babu et al., 2011). The classification of
themselves (for example, I-A, II-B, and III-A) Cas1 and Cas 2 genes: Class 1 / Type I, II, III,
(Zhang et al., 2014). These genes associated and U (Makarova and Koonin, 2015). The
with Cas proteins are named as Cas1, 2, 3, and Cas genes are located in Type I (Cas1, Cas2,
4 (Haft et al., 2005). There is a typical operon Cas3, cas4, Cas5, Cas6, Cas7, Cas8), II
organization for Cas type according to bacte- (Cas1, Cas2, Cas4, Cas9), III (Cas1, Cas2,
ria species. The Streptococcus thermophilus Cas5, Cas7, and Cas10), and U (Cas5, Cas7,
has got three Cas genes as Cas9, Cas1, and Cas8) with the other genes (Csn2, RNAse III,
Cas 2 and Escherichia coli K12 has got six SS) (Makarova et al., 2013) (Table 1).
Cas genes as Cas3, Cas7, Cas5, Cas6, Cas4,
Table 1: Organization of CRISPR-Cas types
Class Types Subtypes Genes Targets
Class Type I I-A, I-B, I- Cas1, Cas2, Cas3, I-A: Archaeoglobus fulgidus
1 C, I-D, I-E, Cas3, Cas4, Cas5, I-B: Clostridium kluyveri
I-F, I-U Cas6, Cas7, Cas8/LS I-C: Bacillus halodurans
I-D: Cyanothece sp.
I-E: Escherichia coli K12
I-F: Yersinia pseudo-tuberculosis
I-U: Geobacter sulfurreducens
Type III-A, III-B, Cas1, Cas2, Cas5, III-A: Staphylococcus epidermidis
III III-C, III-D Cas6, Cas7, Cas9, III-B: Pyrococcus furiosus
Cas10, SS III-C: Methanothermobacter thermauto-
trophicus
III-D: Synechocystis sp.
Type Cas1, Cas2, Cas5, IV: Thioalkalivibrio sp. K90mix.
IV Cas6, Cas7, SS
Class Type II II-A, II-B, Cas1, Cas2, Cas4, II-A: Streptococcus thermophilus
2 II-C Cas9, Rnase III, Csn II-B: Legionella pneumophila str. Paris
II-C: Neisseria lactamica 020-06
Type V-A, V-B, Cas1, Cas2, Cas4, V-A: Francisella cf. novicida Fx1
V V-C, V-D, Cas12 V-B: Alicyclobacillus acidoterrestris
V-E V-C: Oleiphilus sp.
V-D: Bacterium CG09_39_24
V-E: Deltaproteobacteria bacterium
Type VI-A, VI-C, Cas1, Cas2, Cas13 VI-A: Leptotrichia shahii
VI VI-B1, VI- VI-C: Fusobacterium prefoetens
B2 VI-B1: Prevotella buccae
VI-B2: Bergeyella zoohelcum
23EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
These proteins are responsible for the on Cas3, involving CRISPR-Cas3 and human
ability of the CRISPR-mediated immune sys- embryonic stem cell line in HAP 1 (Hunting-
tem in bacteria to adapt to new viral infections ton Associated Protein 1), base sequences in
(He et al., 2018). Type II CRISPR-Cas sys- DNA were deleted rather than cleavage (100
tems use a double target mechanism that in- kilobases (kb)). Thus, some technical prob-
cludes RNA-DNA to eliminate invading path- lems were overcome using Cas9. CRISPR-
ogens. Thus, it checks the strength of CRISPR Cas3 enables the detection of non-coding
immunity. The distinction between the self gene locations in the genome by screening
and the dynamics of Cas10, Type III gene elements that are effective in cell differ-
CRISPR-Cas immunity governs the discrimi- entiation, cancer, and protein expression
nation between self and extra-self in non-self (Dolan et al., 2019). As well as being benefi-
(Wang et al., 2019a). To better understand the cial, it can cause the deletion of the virulence
CRISPR mechanism among these systems, it properties of pathogens due to viral diseases.
is necessary to grasp the importance of the One advantage of working with Cas3 is that it
Cas 9 protein. CRISPR-Cas is the adaptive can successfully correct deletions and inser-
antivirus immune system found in many bac- tions in human cells. A study for the Cas3 pro-
teria. The system injects the foreign DNA tein that deletes a large part of the DNA
fragments into the CRISPR cassettes, then makes this protein different from the others.
copies the CRISPR sequences containing the Furthermore, the use of Cas3 provided more
spacers and processes them to make a guide efficient editing than Cas9 without off-target
CRISPR RNA specifically trying to target and effects. The DMD gene was repaired with
cleave the genome of the cognate virus or Cas3 in induced pluripotent stem cells
plasmid. The importance of Cas proteins (Morisaka et al., 2019). Because of this fea-
emerges here. Because these proteins are ture, Cas3 is a better alternative than other
quite diverse and are required for different Cas proteins in its class. Moreover, setting
stages of processing transcripts of CRISPR drug targets can have an important potential
loci, such as cleavage of target DNA or RNA, in the field of disease prevention and agricul-
integration of new spacers (Barrangou, 2013; ture.
Wiedenheft et al., 2012).
Cas4: Fast virus destroyer that creates mem-
Cas1 and Cas2: Required in the adaptation ories of invading viral elements
process of the CRISPR - Cas system The next Cas protein Cas4 has a role of
Cas1 and Cas2 are two proteins in the pro- specific integration on CRISPR spacers. Stud-
karyotic immune system. Cas1 is an endonu- ies to reveal the importance of Cas4 has
clease that enables the formation of dsDNA shown that bacteria cannot create memory
fragments. Cas2 is used to buy spacers in the when Cas4 is not present (Zhang et al., 2019).
CRISPR system and forms a stable complex Cas4 helps to create memories of invading vi-
with Cas1 and mediates spacer acquisition ral elements, thus protecting the bacterial cell
during CRISPR-Cas adaptive immunity from virus infection. The system quickly
(Wang et al., 2019b). finds and destroys the invader virus in virtue
Cas3: Better genome editing tool than Cas9 of these memories. In the absence of Cas4, the
Another Cas protein is Cas3, and this Cas bacteria create an invader memory but cannot
protein participates in the CRISPR interven- preserve these memories. The reason Cas4
tion in the third stage of CRISPR immunity cannot preserve a memory is because of short
(He et al., 2020) and is essential for phage de- DNA sequences made up of a small number
fense in the CRISPR-Cas system (Jackson et of base pairs that serve as a recognition point
al., 2014). While scientific research on the for proteins such as PAM (Kieper et al.,
CRISPR-Cas9 system continues, the im- 2018). This result also shows that choosing a
portance of Cas proteins continues to be dis- PAM for successful gene editing is crucial.
covered. In a new gene-editing method based
24EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
Cas5-9: Defence response to virus and the target locus (Askjaer et al., 2014). Cas9 is
maintenance of CRISPR repeat elements a molecule that has problems as well as mag-
The Cas5 and Cas6 core genes are two of nificence. Its complementarity is limited and
the genes encoding proteins specific to each sometimes it can cut non-specific DNA se-
of the CRISPR systems (Haft et al., 2005). quences besides the normal cutting function
When Cas5 is catalytically active, it acts as a (Lino et al., 2018). This non-specific cutting
replacement for Cas6. When it is not active, it function can cause serious problems in thera-
can take place in interference and adaptation peutic applications and undesirable conse-
phases. Cas6 and 7 proteins, the product of quences in experimental interventions. Be-
these genes, belong to the repeat-associated sides, its large size makes it difficult to enter
mysterious proteins (RAMP) superfamily and cells containing viral vectors (Xu et al., 2019).
have a sequence or structure-specific RNAse Finally, the rules required in the PAM se-
activity involved in the processing of pre- quence limit the manipulation of the target
crRNA transcripts (Makarova et al., 2011). DNA (Gleditzsch et al., 2019). Which is the
RAMPs are a class of RNA binding proteins. correct Cas9? At this point, Cas9 variants and
These proteins play a role in host immunity. alternatives offer various solutions. The most
They carry a similar sequence in their DNA popular Cas variant is isolated from the bac-
versus the sequences of viral invaders. These terium Streptococcus pyogenes (SpCas9) (Le
sequences in the host DNA selectively de- Rhun et al., 2019). The other Cas types are re-
stroy the virus nucleic acid and provide im- spectively dead Cas9 (dCAs9), Staphylococ-
munity to itself with an RNA-based strategy. cus aureus Cas9 (SaCas9), Streptococcus
RAMP proteins are involved in this defense thermophilus Cas9 (StCAs9), Neisseria men-
mechanism (Wang and Li, 2012). ingitides Cas9 (NmCAs9), Francisella novi-
Cas6-9 proteins are involved in the im- cida Cas9 (NvCas9) (Cebrian-Serrano and
mune systems of various bacteria. These Cas Davies, 2017). What makes them different is
proteins are called cascade-like complex and the variety of PAM sequences they need and
protect crRNA (Brendel et al., 2014). Cas7 is some are small enough to be easily packaged
effective at interference, binds crRNA and it into viral vectors, such as in SaCas9 (less than
may be effective in RNA-guided RNA cleav- 1 kb) (Tsang, 2017). Two new Cas9 variants
age. Cas8 binds DNA and interacts with a have been discovered, recently different from
crRNA loaded RAMP. Thus, it can take part known systems: CasX and CasY. CasX,
in both interference and spacer selection which consists of only 980 amino acids and is
(Makarova et al., 2011). found in bacteria not included in the human
Today, the most widely used technology microbiome. CasX is similar to Cas9 but a
is the CRISPR-Cas9 system. Cas9 is a protein very small protein. CasX can be advantageous
that plays an important role in the immune when our goal is to send small parts to the cell.
system of certain bacteria and is an RNA- Similar to other Cas enzymes, CasX can cut
guided DNA endonuclease enzyme. Bacteria dsDNA accompanied by sgRNA, bind to
use Cas9 to create a memory, interrogate, and DNA for gene control, and target specific
distinguish foreign DNA such as bacterio- DNA sequences. But it has its unique RNA
phage or plasmid DNA (Heler et al., 2015). that performed the same functions (Burstein
The CRISPR system has two basic factors: et al., 2017). The CRISPR-CasY system was
the guide RNA and the nuclease. The guide first discovered in organisms with a small ge-
RNA (gRNA) is complementary to the spe- nome, called Candidate Phyla Radiation
cific DNA sequence. The nuclease is a molec- (CPR), that rarely encoded CRISPR-Cas sys-
ular scissor to separate target DNA. The tems for phage defense and contains a large
gRNA directs Cas9 to its target DNA se- bacterial group (Chen et al., 2019). They are
quence. PAM sequence is needed for Cas9's few in a number, and little information is
cutting function. Cas9 recognizes the PAM available on their function. CasY is also
sequence and creates a double-strand break at
25EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
known as CS12d. Cas12d is the class II effec- and defense against foreign genetic material
tor protein. It binds to dsDNA targets and as viral genomes. In other words, this sug-
cleaves these targets. Unlike CasX, other gests that Cas proteins may play a role in the
CRISPR-based editors are larger and come uptake of new spacers, as well as their effects
from bacteria that infect humans. Another re- on the combination of the effector complex,
markable feature of CasX besides its small depending on the type of Cas in the CRISPR-
size is that it is found in bacteria that humans Cas system.
have never been exposed to. Is this an ad- Cas12 and Cas13: Affect gene expression
vantage or a disadvantage for genome edit- without changing the genome sequence for
ing? Cas9 proteins used for genome editing therapeutic application
originate from bacteria in the human body and Two of the most studied Cas proteins after
carry an immunity memory for Cas9. For Cas9 are 12 (Cpf1-Cas12a) and 13 (Swarts
Cas9, it is not clear what will happen about and Jinek, 2018). Cas12 is a class 2, type V
the behavior of the immune system in a per- effector protein (Makarova et al., 2020), and
son with this memory. Can it be preferable to is an effective enzyme that produces cascade
include CasX and CasY in non-human bacte- cleavage in dsDNA. It is used as a platform
ria for gene therapies to be made in humans? for the regulation of the epigenome as it can
They are questions that researchers need to process its guide RNA. It is also known that
find answers to. Cas12a can arbitrarily cleave single-stranded
Cas10 and Cas11: DNA binding, endodeoxy- DNA after it has been activated by a target
ribonuclease activity DNA molecule that matches the spacer se-
The CRISPR-Cas10 system differs from quence (Chen et al., 2018). In this way,
other CRISPR systems because it does not re- Cas12a can detect a small amount of DNA in
quire a PAM sequence and can identify se- a mixture and uses an RNA molecule as a
quences even in the presence of point muta- guide to find the complementary DNA se-
tions (Bari et al., 2017). CRISPR-Cas10 sys- quence. Cas12 cuts both strands of the target
tem is a potential candidate for gene editing DNA it determines (Rusk, 2019). The cutting
due to these features. Cas11 is a subunit in process initiates the repair process that causes
type I and III (Dorsey et al., 2019; Majumdar changes or editing in the genomic DNA se-
and Terns, 2019; Shmakov et al., 2018) effec- quence (Yao et al., 2018). Cas proteins target-
tor complex and involves in the maintenance ing RNA are promising for the antiviral ap-
of CRISPR repeat elements (Majumdar and proach in treatment. Cas13 is a class II and
Terns, 2019). There are a limited number of type VI protein (Terns, 2018). It effectively
studies about the basic functions of Cas11. targets and cleaves of RNA in mammalian
Classification in the CRISPR-Cas system in- cells (Abudayyeh et al., 2017) and various
cludes effector modules in class 1 systems. model systems (Pickar-Oliver and Gersbach,
These modules consist of multiple Cas pro- 2019). Today, effective and cost-effective so-
teins. Some of these are in the form of lutions are needed for the diagnosis of dis-
crRNA-binding complexes (cascade complex eases. Synthetic biomolecules are used as a
1), and this form mediates pre-crRNA pro- solution. However, it may not be possible to
cessing and interaction with the contribution provide all of their specificities, sensitivity,
of additional Cas proteins. As described speed, cost and convenience together, but it
herein, Cas11 is a member of effector mod- may not be possible to provide all their speci-
ules and is involved in crRNA processing and ficity, sensitivity, speed, cost, and conven-
interference. The interference or effector ience together. Recently, a Specific High-
module is involved in target recognition and Sensitivity Enzymatic Reporter UnLOCKing
nucleic acid cleavage. Additionally, the effec- (SHERLOCK) complex was created with an
tor modules have different combinations of RNA-guided and RNA-targeted CRISPR-
Cas protein (Shmakov et al., 2018). The pur- Cas13a. SHERLOCK has high sensitivity and
pose of these modules is to target surveillance specificity and is very suitable for detecting
26EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
target RNA and is a CRISPR based diagnos- based systems such as Ebola, Zika, Lassa
tic. However, it is unsuitable for detecting (Myhrvold et al., 2018) and COVID-19 (Bai
DNA sequences, and in vitro transcription of et al., 2020a) that allow detection in the event
DNA to RNA must be performed before the of an epidemia. The results are promising. Be-
SHERLOCK test. In the study conducted to cause instead of using the CRISPR applica-
solve this problem, CAS12a/crRN/ target tion that requires training, this method ena-
DNA triple complex was created. By adding bles laboratory personnel to obtain viruses di-
a quenched fluorescent ssDNA reporter to this rectly from biological samples such as blood
system, an Hour Low-Cost Multipurpose and saliva and to conclude them quickly. In
High-Efficiency System (HOLMES) was de- this way, cost, time and equipment is saved.
veloped. The system can be used for the rapid Cas13 is the only known prokaryotic
detection of target DNA as well as target CRISPR-Cas system that targets the single
RNA (Li et al., 2018). It should not be over- RNA. Therefore, RNA has more places in
looked that the CRISPR system has deficien- specific applications and is preferred over
cies as well as its benefits. As with other di- other Cas types for targeted RNA degradation
agnostics, this system is generally not perfect. and gene knockdown, RNA editing, nucleic
However, its applicability is easier and faster acid detection, and patient diagnosis (Terns,
than previous gene therapy systems. It is a 2018).
great advantage that the system has a high ca- Cas14: Benefit and loss accounting
pacity to accept accessory molecules and can compared to Cas9, 12 and 13
be shaped according to the treatment goal. Cas14 is also advantageous in biotechno-
Cas13 has CRISPR array processing effi- logical applications. Cas14 is an RNA-guided
ciency and is ideal for multi-target applica- nuclease and can provide targeted ssDNA
tions. Most importantly, it is widely used in cleavage without requiring restrictive se-
biotechnology and clinical applications. It is quences. An important detail is that Cas14
possible to specifically identify targeted DNA does not require a PAM sequence and recog-
or RNA sequences using Cas13 (SHER- nizes ssDNA more specifically than Cas12
LOCK) (Kellner et al., 2019). The clinical and 13. Cas14 is a newly discovered Cas pro-
significance of this system is that RNA or
tein. Although Cas14 is smaller than Cas9, it
DNA can be detected multiplexed and sensi- can target ssDNA in defense against viruses
tively in relevant samples. When we look at with the ssDNA genome. Owing to this fea-
the features of this system, we see that we can ture, Doudna and her team combined Cas14
target mammalian viruses and thus create an with the isothermal amplification method
antiviral platform. This platform allows in- called DNA endonuclease-targeted CRISPR
vestigation of the prevalence of target regions trans reporter (DETECTR-Cas14)
in viral genomes containing ssRNA and thus (Harrington et al., 2018). Cas14 has a big ad-
enables the identification of possible targets. vantage due to its small size. Cas14 can pro-
However, there is still a need to develop strat- vide regulation in both cells and viral genes
egies to determine whether the Cas13 activity and contribute to the development of
is being performed correctly and to optimize CRISPR-based diagnostic systems when it
this behavior while scanning these targets. comes to infectious diseases, mutations, and
The SHERLOCK system has been used in cancer with ssDNA cutting activity. Cas9, 12,
some patient samples to detect different vi- 13 and 14 are in a competition that can vary
ruses (Myhrvold et al., 2018). The system depending on the single or dsDNA or RNA
searches for specific nucleic acids, and test they target. It would be wrong to say which of
strips are used to make them visible. The pres- these is the best. Cas12 is successful in recog-
ence of the virus is determined by the line that nition of dsDNA, recognition of Cas13
will be formed after the paper strip is dipped ssRNA and finally recognition of ssDNA of
into the sample to be examined. Using these Cas14. It is understood that Cas's alone are
systems, researchers are working on CRISPR-
27EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
enough to fulfill their functions. Can SHER- CRISPR BEHAVIOR IN BIOLOGICAL
LOCK and DETECTR be combined? These SYSTEMS
techniques are diagnostic tools that can be Researchers have focused on solving cur-
used to specifically detect low concentrations rent problems such as human diseases, food
of RNA and DNA. Heating Unextracted Di- quality and productivity by developing
agnostic Samples to Obliterate Nuclease CRISPR technologies. Human diseases take
(HUDSON) technique is used to directly de- the first place among the purposes of CRISPR
tect nucleotides in body fluids. When HUD- applications. The most easily treatable group
SON and SHERLOCK are used together, they is mutational diseases with a direct relation-
can be combined to detect RNA and DNA in ship between the cause of the disease and the
a small amount of sample urine, saliva, serum, clinic. One way of treating diseases using
plasma and whole blood (Gronowski, 2018). these methods is to mimic the disease by mod-
This system allows the editing of mutations eling it in cell, model organisms, or transgenic
and genes, rapid identification of viral and animals, due to ethical concerns in human
bacterial pathogens, and pathogen-resistant studies. In addition, they can be implanted
genes in the same analysis. Aiming to in- into cells by plasmid transfection to model
crease the target specification of CRISPR- cellular defense in infectious diseases and can
Cas9, researchers focused on Cas9, 12, 13 and be used to create disease models at the cellular
14. These 3 proteins have different ad- level. With these techniques, we can edit the
vantages as diagnostic. Cas proteins must DNA of organisms, delete genes at any stage
meet certain criteria. Also, the character of the of its formation, and even the entire chromo-
nucleic acid sequence the user wants to detect some. This deletion procedure can be useful
is important. Specific nucleic acid sequences in the treatment of extrachromosomal disor-
to be detected are cut with protein guide mol- ders such as Down syndrome and Triple X
ecule complexes. The advantage of these (Cowan et al., 2019). Today, CRISPR-Cas
complexes is that they do not specifically cut technology is used in diseases such as cystic
other nucleic acids after the target sequence fibrosis, hemophilia, DMD, sickle-cell ane-
has been cut. When the cutting process is fin- mia, -thalassemia, cancer, and Huntington's
ished, they generate a visual signal along with disease. Research continues for MHC pro-
dCas9 labeled with an enhanced green fluo- teins, which are important for tissue engineer-
rescent protein (eGFP). Only user-specified ing and stem cell engineering, regenerative
nucleic acids are truncated (Zhou et al., 2018). medicine (Hsu et al., 2019), and tissue and or-
The aim is a fast and reliable diagnostic com- gan rejection (Abrahimi et al., 2016). These
bination. The key point of these systems is the initiatives predict that CRISPR will find more
type of Cas to be used. The advantages or dis- places in our lives in the future. As such, these
advantages of the system, depending on the technologies seem important not only in gene
Cas's, depending on the length of the protein, editing but also in synthetic biology. The pur-
the size of the single guide molecule or the ac- pose of synthetic biology is to create bio-com-
curacy of the process and the nature of the tar- puters, to produce genetic circuits that work
get to be manipulated. and have memory. Thus, it is planned to pro-
The main criteria to be considered in the gram mammalian cells using Cas9 variants,
use of the system include the type of Cas pro- which are protein-based processors that con-
tein and system to be used (HOLMES, SHER- trol gene expression in response to single-cell
LOCK, HUDSON-SHERLOCK, DETECTR, RNAs (Kim et al., 2019). Is it possible to rec-
SHERLOCK-DETECTR), effector molecule, ord on DNA using this data? To store the data
sensitivity, specificity, fluorescent molecule, in DNA, the researchers created an empty re-
type of the target (DNA, RNA, protein) and cording space that mimics the "0" and "1" sys-
duration (Kostyusheva et al., 2020; Li et al., tem in the computer. Then, based on the 4 ba-
2019). ses in DNA, they evaluated "A and C" as 0,
"T and G" as 1, according to the Watson-
28EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
Crick pairing, and found that these data were through the population and repairs the sensi-
correctly translated by the system. As a result, tive allele by encountering the resistant allele.
this system was envisioned as a natural mech- The most important observation of the study
anism by which a copy of DNA is created is that errors caused by allelic drivers are not
(Church et al., 2012). With the CRISPR sys- transferred to the next generation and as a
tem, it may be possible to store data in DNA. thought-provoking result, an extraordinary
The creation of this code, which will be re- situation called "fatal mosaic" emerged. It is
flected in the entire living system, may have thought that this can be used as an advantage
critical consequences for the organism. How- to eliminate unwanted mutations caused by
ever, it seems possible and controllable to cre- drivers containing CRISPR and make them
ate this software in a single cell. Recently, the more efficient. Based on this application, in
CRISPR gene editing system was applied for order to correct the faulty allele with the
the first time in one patient. In this procedure, CRISPR-Cas9 system in human diseases, this
white blood cells of two patients with multi- faulty allele must be enzymatically cut and
ple myeloma and sarcoma were obtained. corrected by replicating the error with an ex-
These cells were arranged with the CRISPR ogenous oligonucleotide or dsDNA template.
system and reintroduced to the patient In a study on this subject, it is aimed to show
(Stadtmauer et al., 2020). To develop anti- that it is possible to repair the other allele to
tumor immunity in patients' T lymphocytes, be sensitive to cutting by creating a cut-re-
the CRISPR technique was applied to TRAC, sistant allele for heterozygous individuals in
TRBC, and PDCD1 genes and New York the germ line (Guichard et al., 2019). This
esophageal squamous-cell carcinoma 1 (NY- model can be applied in animal models and
ESO-1) was used to recognize tumor cells as cell lines to mimic many human diseases. Alt-
well-known cancer-testicular antigen hough allelic correction and intergenerational
(Thomas et al., 2018). As a result, it was ob- transfer seem to be good practice, as stated be-
served that patients could tolerate this gene fore, unexpected results may be encountered
therapy. The results regarding the long-term due to the uncontrollable features of the
effectiveness of the application, the patient's CRISPR-Cas9 system. Another study on this
life and recovery time, or the relapse of the subject is the deletion of the memory of rats
disease are closely related to the individual re- with CRISPR (Sun et al., 2020). This im-
sponses of the patients, and the success of the portant claim will pave the way for the
application depends on many factors. Treat- CRISPR system, to which we refer to rewrit-
ment approaches are promising. What would able codes, to erase and restore memory and
happen if this change was passed on to future perhaps load data that will at least restore peo-
generations? Gene drivers created using the ple's daily routines in diseases such as Alzhei-
CRISPR system makes it possible to design mer's. However, considering that memory is
certain features that can be transferred be- not alone and epigenetic memory is effective
tween generations. For this purpose, the al- in emotions, situations, and behaviors, it is
lelic drive system equipped with guide RNA, still uncertain how the transfer of an epige-
which allows replacing the unwanted variant netic molecule pool to individuals with
of a gene with its preferred variation, has been CRISPR intervention will have conse-
developed. Desired genetic features such as quences. At this point, organisms that inspire
drought resistance, increased crop yield, re- genetics are very important. Understanding
sistance to pests, sensitivity to pesticides can these organisms enables the determination of
be spread to populations or products can be treatment options in humans by creating ap-
given resistance to mutagens. This study propriate models. As a fundamental mecha-
shows that insects are a good source for allele nism, a major change in DNA is transmitted
drivers (Guichard et al., 2019). By adding the by the mRNA from the nucleus to the cyto-
engineered gRNA to a gene driver, it was plasm. If RNAs are arranged outside of the
found that the allele gene driver spreads
29EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
nucleus, what would be the result? The an- The cellular security provided CRISPR
swer is given by a group of researchers on the system can be used to turn genes on or off.
Doryteuthis pealeii (longfin inshore squid). This initiative could allow genes to be man-
Researchers reported that this cephalopod aged when, where and for how long. In a new
was the first known animal to regulate mes- method on this topic, CRISPR gene editing
senger RNA outside the cell nucleus enzymes were injected directly into the brain
(Vallecillo-Viejo et al., 2020). Permanent of a developing mouse fetus as a therapeutic
changes in DNA make rearrangement diffi- intervention (https://www.spec-
cult to do here. However, when using mRNA, trumnews.org/news/injecting-crispr-fetal-
it is possible to avoid treatment-induced er- brain-may-correct-autism-mutations/). The
rors due to its half-life. When an RNA editing study is based on the opening of the UBE3A
similar to that of squid is made in the cyto- gene before birth, which causes Angelman
plasm, if the result is outside what is expected, syndrome as a result of a mutation in the de-
the regulation can be done by replacing it with veloping brain. The results of the study con-
an analog. In this way, cytoplasmic correction ducted in 10 mice show that the gene reac-
of mutations will pave the way for personal tivated using CRISPR at the age of five
treatment in humans. Editing between the nu- months after treatment works in about half of
cleus and the mitochondrial genome, can be the neurons in the brains of mice. As in these
fatal to mitochondria due to the lack of a mi- studies, genes can be silenced or activated
tochondrial repair mechanism. The sequence with CRISPR technology. The application of
of the mRNAs, the proteins they encode and the procedure at the developmental or stem
the information they carry can be changed by cell stage is sensitive, and it is important to
intervening in the plan of the genome without which part of the animal it will be performed
disrupting them. To this end, the researchers (tail, head or brain). Another interesting
demonstrated that genetic information can be CRISPR application was discovered by re-
re-encoded in a region-specific manner in the searchers working on pain. The subject of the
regulation of neuron RNA via the ADAR2 en- study is an antidote containing CRISPR tech-
zyme, which is expressed outside the nucleus nology that reduces pain caused by Australian
of squid neurons. In mammals, this enzyme is venomous jellyfish venom (Lau et al., 2019).
both diverse and largely expressed in the nu- The genome editing technique used for this
cleus and nucleolus. This mechanism in squid deadly venom can improve symptoms 15
can be mimicked by evaluating its molecular minutes after exposure. Can pain be treated
structure, diversity and cellular location and with this method in human pain panel and
can be applied in human cells and animal chronic diseases? There is a wide spectrum
models. It is stated that such a control can be from migraine to rheumatic pains. The tech-
achieved with small-molecule inhibitors of nology subject to the research was applied by
Streptococcus pyogenes Cas9 (SpCas9) pro- the injection method. The aim of the research-
tein (Maji et al., 2019). The biggest concern ers is to develop the form that can be applied
in this application is in reducing the off-target to the skin. At this point, the problem we al-
effects of Cas9. In solving this problem, it ways face is that CRISPR has off-target ef-
may be useful to design and use small-mole- fects by cutting the wrong piece of DNA. The
cule inhibitors (anti-CRISPR) for CRISPR- technology applied is similar to the use of
related nuclease using computational biology. stem-loop primers in in vitro analysis of short
The problem with existing anti-CRISPR mol- sequence miRNAs. In the method, in addition
ecules is that they are large and cannot enter to the CRISPR mechanism, a short tail of 20
the cell. Small-molecule inhibitors are favor- nucleotides locked to the gRNA was added.
able because they are recyclable and are not When the tail reaches the target RNA, it folds
degraded by cellular proteases and immune back and binds to the RNA, forming a hairpin
system reactions. that allows it to cut Cas9 exactly at that site.
30EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
As new molecules and applications were dis- Morris, 2018). This change is important. Be-
covered, tests performed on different CRISPR cause many of the known diseases can be cor-
systems involving human cells show that gene rected using this change. Hurler syndrome is
editing accuracy has increased (Kocak et al., one of them and a rare lysosomal storage dis-
2019). Can we solve every problem we en- ease. To treat this syndrome, a ZFN-mediated
counter with CRISPR? Looking at the re- gene editing study was performed using the
searchers, publications and the presence of murine model, and a correct copy of the
numerous opened research companies, espe- IDUA gene was inserted at the albumin locus
cially the recent COVID outbreaks have led in hepatocytes. Thus, this metabolic disease
us to turn to this technology, which has the was corrected by providing continuous ex-
most common and easily applicable potential. pression of the enzyme (Ou et al., 2019). As
As a result, the CRISPR-Cas system is an ef- shown by this study, LEAPER is preferred in
fective and promising technology for the to- similar studies because it is simpler than ex-
day and future. isting techniques. The distinguishing feature
of LEAPER from other techniques is that
LEAPER CRISPR requires components such as gRNA
The changes in the nature of CRISPR and Cas enzyme, whereas the only aRNA is
technology have led to the search for new al- used in this technique. Thus, a gene-editing
ternatives. One of these changes is the devel- alternative is provided with low immune re-
opment of LEAPER technology, which is jection and easy delivery to the target. In this
thought to be safer and has fewer side effects study, it is aimed to restore the catalytic activ-
by targeting RNA instead of DNA (Qu et al., ity of α-1-iduronidase (IDUA) in the primary
2019). This technique includes endogenous fibroblasts of patients. The promises of tech-
ADAR (ADAR1 and ADAR2) and native nologies to correct genetic errors are striking,
proteins. Thus, immunogenic reactions can be but the problems of these technologies cannot
prevented. The technique requires the use of always be tolerated by the editing system or
specially engineered RNAs (aRNAs-ADAR the elements in the system. Abnormal regula-
RNAs) that combine naturally-occurring en- tion of RNAs in multiple myeloma where
zymes to convert specific adenosine to ino- ADAR1 is overexpressed leads to off-target
sine. While this application is successful in regulation by increasing oncogenicity. More-
cellular studies, research continues in rats. over, ectopic expression of proteins is a po-
Recently, a cytidine and adenosine deaminase tential risk for immunogenicity. Because the
has been used to correct disease-causing mu- adaptive immunity in the cell and the DNA
tations. In this method, DNA base editors repair response involving p53 may compro-
were constructed, and ADAR adenosine de- mise the efficacy of a therapeutic protein such
aminases were used to precisely regulate as Cas9. However, some studies favor ADAR
RNAs to adenosine-inosine (A-to-I) and com- because it does not have immune-stimulating
bined with CRISPR-Cas9 (Qu et al., 2019). effects and does not interfere with the func-
A-I editors have a broad spectrum and can af- tion of endogenous ADAR proteins. When
fect major mechanisms that can affect gene engineered ADARs for RNA editing are ex-
expression, such as modification of protein- amined, apart from the A-to-I editors men-
coding sequences, splice site modification, tioned so far, there are also C-to-U editors,
RNA's ability to modify itself against nuclear although they are not often mentioned (Fry et
exposures, microRNA sequences, and their al., 2020). A deaminase has been developed
target regions (Quinones-Valdez et al., 2019). that allows the regulation of cytosine and ura-
Three types of ADAR proteins have been cil in RNA by mutagenesis of ADAR2. The
identified, the substrate of which is a double disease in which ADAR technology is applied
chain RNAs. During translation, inosine is is hereditary retinal degeneration. In this dis-
thought to mimic guanosine (Shevchenko and ease, blindness occurs as a result of the death
and dysfunction of outer retinal cells due to
31EXCLI Journal 2021;20:19-45 – ISSN 1611-2156
Received: October 22, 2020, accepted: December 10, 2020, published: January 04, 2021
mutations in the heterogeneous gene se- molecular elements such as metric computa-
quence. Researchers hope that this treatment tion, coding, non-coding and intergenic re-
could stop or reverse vision loss in patients. gions, repeating elements, telomeres, and
LEAPER is preferred because it is a target- transposons can be calculated. This computa-
specific, reversible technology that does not tion can help us analyze RNA folds with
have low immune reactions, does not have the LEAPER technology and discover the abili-
risk of creating permanent non-target muta- ties of regulatory elements and non-coding
tions. Also, LEAPER technology has been RNA (Andrews et al., 2017). Because RNA
used to correct pathogenic G>A change in folding errors must be known to be able to
cells (Abudayyeh et al., 2019). One of the edit. A study reveals the importance of this
therapeutic targets of LEAPER is the tumor idea. The study demonstrates the complex and
suppressor gene TP53 (non-functional trun- regulatory role of ADAR1, which forms the
cated protein is formed) (Floquet et al., 2011). basis of LEAPER technology, on RNA sec-
The gene is primarily responsible for main- ondary structure (Solomon et al., 2017). Er-
taining cellular homeostasis, and the fre- rors due to re-expansion of microsatellites due
quency of mutations in the gene is high in to RNA secondary structure are generally
more than 50 % of human cancers. This study seen as myotonic dystrophy types 1 and 2,
demonstrated that LEAPER repairs the early Fragile-X, amyotrophic lateral sclerosis, fron-
stop codon of TP53 and restores its function. totemporal dementia, and tautopathies
The ability of aRNAs to be efficiently deliv- (Bernat and Disney, 2015). If A-I editing is
ered to the cell as vectors or as a synthetic ol- done, the structure and base pairing property
igonucleotide is promising for therapeutic ap- of the RNA molecule may change. This
plications with high regulatory activity. The change is important in controlling the number
application spectrum of this technology is of functional RNA molecules in the cell and
wide. One of these is miRNAs and they are a providing cell defense (Wulff and Nishikura,
good resource because of their therapeutic po- 2010). A-I editing can be used as a system
tential. MiRNAs have multiple targets, and with RNA basic regulators consisting of
multiple miRNAs are specific to a single tar- Cas13 and ADAR. Thus, higher editing effi-
get. In addition to these features, the seed se- ciency can be achieved by targeting more spe-
quences of miRNAs are suitable for editing as cific. It is important to consider ethical rules
they allow A to I regulation. miRNAs are in researches on human and other living life.
translationally repressors and affect gene ex- Before genome editing becomes an interven-
pression. Given that many genes are regulated tion with unpredictable results, initiatives that
by miRNAs, mapping hotspots for editing can can control individual responses and reduce
be a guide for treatments such as cancer ethical concerns should be included, espe-
(Wang et al., 2017). With this technology, cially in human applications.
diseases can be treated by changing the main
targets of irregular miRNAs (van der Kwast RESTORE
et al., 2018). Regulation in the miRNA seed Today, the aim of gene editing technolo-
region determines mRNA selection and gene gies is to restore hotspots needed in the ge-
silencing activity (Kume et al., 2014). More- netic basis of organisms. RNA editing can be
over, this regulation allows interfering with a reliable and effective alternative to gene ed-
RNA metabolism, involving the folding, pro- iting in clinically specific situations. Another
cessing, localization, and degradation of RNA application of ADAR is based on the control
(Hong et al., 2015). Since RNA folding also of exogenous RNA-regulating enzymes or
affects the biology of the molecule, the fold- overexpressed endogenous ADAR enzymes.
ing pattern and secondary structure properties This method involves the use of chemically
can be revealed using databases. Using these optimized antisense oligonucleotides that call
databases, it contains folding information for ADARs to the task of editing endogenous
each base pair, and the folding properties of
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