G C A T

T A C G
G C A T
genes
Review
CRISPR/Cas9 Genome-Editing Technology and Potential
Clinical Application in Gastric Cancer
Renata Sanches Almeida 1 , Fernanda Wisnieski 1,2 , Bruno Takao Real Karia 1 and
Marilia Arruda Cardoso Smith 1, *

1 Discipline of Genetics, Department of Morphology and Genetics, Federal University of São Paulo,
Rua Botucatu, 740, São Paulo 04023900, Brazil
2 Discipline of Gastroenterology, Department of Medicine, Federal University of São Paulo, Rua Loefgreen,
1726, São Paulo 04040002, Brazil
* Correspondence: [email protected]; Tel.: +55-11-5576-4848 (ext. 2378)

Abstract: Gastric cancer is the subject of clinical and basic studies due to its high incidence and
mortality rates worldwide. Due to the diagnosis occurring in advanced stages and the classic
treatment methodologies such as gastrectomy and chemotherapy, they are extremely aggressive and
limit the quality of life of these patients. CRISPR/Cas9 is a tool that allows gene editing and has
been used to explore the functions of genes related to gastric cancer, in addition to being used in
the treatment of this neoplasm, greatly increasing our understanding of cancer genomics. In this
mini-review, we seek the current status of the CRISPR/Cas9 gene-editing technology in gastric cancer
research and clinical research.

Keywords: gastric cancer; CRISPR; gene editing

Citation: Almeida, R.S.; Wisnieski, F.;

Takao Real Karia, B.; Smith, M.A.C. 1. Introduction
CRISPR/Cas9 Genome-Editing
Gastric cancer (GC) remains an important cancer worldwide, ranking fifth for inci-
Technology and Potential Clinical
dence and fourth for mortality globally [1]. The high mortality rates of GC are mainly
Application in Gastric Cancer. Genes
due to its detection at advanced stages, where it can no longer be treated with curative
2022, 13, 2029. https://doi.org/
intent, and to the few available therapeutic options [2]. Currently, gastrectomy is the
10.3390/genes13112029
main option for locoregional GC treatment [3]. However, even after curative resection and
Academic Editors: Xin Liu and adjuvant chemotherapy, the clinical outcome remains poor, with a recurrence rate of approx-
Yihui Fan imately 35% [4]. Despite some recent advances that have been made in the treatment of GC
Received: 20 October 2022
(e.g., systemic chemotherapy, radiotherapy, surgery, immunotherapy, and targeted therapy),
Accepted: 1 November 2022
only a subset of patients can benefit from these treatment options [5].
Published: 4 November 2022
GC is a complex, heterogeneous, and multistep disease that involves environmen-
tal factors, mainly Helicobacter pylori infection, and several molecular alterations, such
Publisher’s Note: MDPI stays neutral
as genetic instability, the inactivation of tumor suppressor genes, and the activation
with regard to jurisdictional claims in
of oncogenes.
published maps and institutional affil-
Moreover, the molecular profiles may vary from patient to patient. Although many
iations.
studies proposed a great number of diagnostic and prognostic markers, only a few of them
are currently used in clinical practice [6]. Furthermore, different epigenetic alterations have
been reported to activate oncogenes and inactivate tumor suppressor genes during gastric
Copyright: © 2022 by the authors.
carcinogenesis [7]. Therefore, an understanding of the molecular aspects involved in gastric
Licensee MDPI, Basel, Switzerland. carcinogenesis and tumor heterogeneity is essential to providing new multidisciplinary
This article is an open access article treatments [8].
distributed under the terms and In this context, gene-editing technology has received increasing attention in GC, as it
conditions of the Creative Commons can adjust gene expression alterations and correct mutations. In addition, this technology
Attribution (CC BY) license (https:// can also reveal the roles of unknown genes in important pathways, and consequently
creativecommons.org/licenses/by/ can help in the identification of new biomarkers and therapeutic targets, as well as the
4.0/). mechanisms of GC responses to drug treatment [9].

Genes 2022, 13, 2029. https://doi.org/10.3390/genes13112029 https://www.mdpi.com/journal/genes

Genes 2022, 13, 2029 2 of 13

Since Kim et. al.’s study, the methodologies for gene editing have involved the use of
the zinc finger nuclease (ZFN) and the transcription activator effector nuclease (TALENs) by
targeting DNA domain-binding proteins [10–14]. Currently, clustered regularly interspaced
short palindromic repeat (CRISPR)-associated 9 is a gene-editing tool with a lower cost,
higher efficiency, and less complexity in its application. In addition, the 2020 Nobel Prize
in Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for the
development of the CRISPR/Cas9 gene-editing technology [15].
In this mini-review, we first describe the mechanism and development of the
CRISPR/Cas9 gene-editing system. Furthermore, we focus on the current applications of
this technique for the basic research, diagnosis, and therapy of GC. The potential applica-
tions of CRISPR/Cas9 in GC therapy and the challenges are discussed.

2. The CRISPR/CAS9 Technology

The first hint of CRISPR was in 1987, when Japanese scientists studied the alkaline
phosphatase gene in Escherichia coli and discovered an adjacent region of unknown func-
tion [16]. After two decades, it was discovered that these previously unknown regions
were related to the adaptive immune systems of bacteria and prokaryotic organisms such
as archaea [17–19]. This finding favored the discovery of the association of the catalytic
enzyme Cas9 with the CRISPR system, providing the double-stranded DNA breaks [20]. In
2013, the CRISPR/Cas9 system was used for the first time as a gene-editing tool [21–23].
CRISPR/Cas9 works with a simplified system using a guide RNA (gRNA) that can
recognize the genomic target through base complementarity according to the Watson–Crick
theory, and also has the function of aggregating the endonuclease Cas9. Due to its simple
system, greater specificity, and efficiency, the CRISPR/Cas9 system is a much more effective
tool than the previous ZFN and TALENs methodologies [24].
The CRISPR methodology has been improved over time (Figure 1), and to determine
the specificity of the machinery connection, a PAM (protospacer-adjacent motif) primer
sequence of 3 nucleotides (NGG) before the 20 nucleotides of the target is determined.
In addition, the Cas type II protein from Streptococcus pyogenes (SpCas9) has been used
for the break of the double strand of DNA. In response to DNA cleavage, the host cell
can perform two different repair mechanisms. The non-homologous end joining (NHEJ)
is a mechanism where the ends of the tape come together, which is subject to unwanted
insertions or deletions. Another mechanism is homology-directed repair 3(HDR),
Genes 2022, 13, x FOR PEER REVIEW of 14 which
uses recombinations already determined from DNA models to reconstitute the cleaved
DNA (Figure 2).

Figure 1. Timeline of CRISPR technology [16–19,21,25–32].

Figure 1. Timeline of CRISPR technology [16–19,21,25–32].
Genes 2022, 13, 2029 3 of 13
Figure 1. Timeline of CRISPR technology [16–19,21,25–32].

Figure 2. CRISPR repair mechanism. Classic CRISPR mechanism comprising the guide RNA (gRNA),
target DNA, and endonuclease (Cas9), which promotes DNA cleavage by DSBs induced by Cas9.
DSBs canFigure 2. CRISPR
be repaired repair
by two main mechanism.
mechanisms: Classic
the NHEJ CRISPRwhere
mechanism, mechanism comprising
the ends of the strands the gui
(gRNA),
come together (thistarget DNA,
is subject and endonuclease
to unwanted (Cas9),
insertions or which
deletions), promotes
or by the HDRDNA cleavage
pathway, whichby DSBs
by Cas9. DSBs
uses the recombination can be
donor DNArepaired by to
templates two main mechanisms:
reconstitute the DSB. the NHEJ mechanism, where th
the strands come together (this is subject to unwanted insertions or deletions), or by the HD
In addition,
way, whichlarge chromosomal
uses deletions,
the recombination inversions,
donor and translocations
DNA templates [33],the
to reconstitute as well
DSB.
as plasmid and retrotransposon insertions [34,35], have also been reported in the literature
as a result of the DSBs induced
In addition, largebychromosomal
CRISPR/Cas9.deletions, inversions, and translocations [33]
Accordingly, the ability of CRISPR/Cas9 to generate DNA double-stranded breaks at
as plasmid and retrotransposon insertions [34,35], have also been reported in the li
sites of interest, introducing a donor DNA template into the target cell, means it has been
as a result of the DSBs induced by CRISPR/Cas9.
proven to be a useful tool for genome editing [36,37].
Moreover, using a modified version of the Cas9 protein, dCas9 (catalytic “dead”
Cas9), it is possible to target desirable regions on the genome without cutting the DNA
strands [25,38], which enables the possibility of adding many different proteins to dCas9,
including fluorescent proteins. In 2013, Chen and collaborators described a method for
the imaging of repetitive elements in telomeres and coding genes in living cells using a
CRISPR/dCas9-EGFP tag [26]. Interestingly, regulatory factors such as modulators of gene
expression can also be fused to dCas9. Like RNAi (RNA interference), CRISPRi (CRISPR
interference) may be used to repress gene expression, although using a different mechanism.
While RNAi uses a nucleotide base complementary to the desired sequence and the RNA-
induced silencing complex (RISC) to suppress an mRNA target [39], CRISPRi uses a dCas9
linked to a transcription repressor domain to identify and bind to a specific DNA locus,
and then to inhibit its transcription [40]. On the other hand, it is also possible to conjugate a
dCas9 to a transcription activator and to create a CRISPR activation (CRISPRa) system [40].
Hilton and colleagues revealed that CRISPR/dCas9 fused to the human acetyltransferase
p300 can be used to activate gene transcription by acetylating histone H3 lysine 27 at its
DNA target sites (Figure 3) [27].
13, x FOR PEER REVIEW 5 of 14
Genes 2022, 13, 2029 4 of 13

Figure 3. Mechanisms of CRISPR, CRISPRa, and CRISPRi: (a) classic CRISPR mechanism compris-
Figure 3. Mechanisms of CRISPR, CRISPRa, and CRISPRi: (a) classic CRISPR mechanism compris-
ing a guide RNA (gRNA), target DNA, and endonuclease (Cas9), which promotes DNA cleavage,
ing a guide RNA (gRNA), target DNA, and endonuclease (Cas9), which promotes DNA cleavage,
resulting in a knockout; (b) CRISPR mechanism comprising a guide RNA (gRNA), sequence target
resulting in a knockout; (b) CRISPR mechanism comprising a guide RNA (gRNA), sequence target
(target
(target DNA), and “dead” DNA), and “dead” endonuclease
endonuclease (dCas9),
(dCas9), without without its
its catalytic catalyticfused
domain, domain, fused
with with inhibitory
inhibitory
transcription factors favoring decreased expression and generating knockdown; (c) CRISPR CRISPR
transcription factors favoring decreased expression and generating knockdown; (c) mech- mech-
anism comprising anism comprising
a guide a guide RNA
RNA (gRNA), (gRNA),
target target
sequence sequence
(target (target
DNA), and DNA),
“dead”andendonuclease
“dead” endonuclease
(dCas9), without its catalytic domain, fused with transcriptional activating
(dCas9), without its catalytic domain, fused with transcriptional activating factors, favoring factors, favoringin-increased
expression and causing upregulation of
creased expression and causing upregulation of a given gene. a given gene.

Additionally, a dCas9 may be engineered to introduce point mutations to a specific

Garcia-Bloj
DNAet al. [43], bywithout
sequence comparing the
causing ZFN,
DSBs. TALENs,
Fusing and CRISPR
a single-stranded technologies
DNA for
deaminase enzyme to
the overexpression of tumor suppressors in GC strains, demonstrated that CRISPR-dCas9
dCas9 allows for single-nucleotide modifications at a genomic sequence of interest. Taking
can restore theadvantage
expression of of the MASPIN/REPRIMO
a segment genes, being
of accessible single-stranded DNA considered
formed by adCas9,
transient,
gRNA, and
targeted, and efficient
target DNA,toolso
forfarrestoring tumor
cytidine and suppressor
adenine functions.
deaminases have been used to convert cytosine
into uracil and adenosine into inosine, respectively [28,29,41].
In the context ofin
3. Application of CRISPR/CAS9 gastric
Basicdiseases, Krishnamurthy and collaborators studied gastric
GC Research
organoids derived from two individuals with homozygous mutations in PDX1 (pancreatic
CRISPR/CAs9 is an extremely
and duodenal homeobox versatile tool that
1), causing canofbeitsused
a loss in the study
expression. and under-
PDX1188delC/188delC
standing of cancer development mechanisms and in the diagnosis and treatment of sev-
organoids resulted in gastritis, a loss of antral identity, and gastric and intestinal metaplasia.
eral diseases, including GCthe
Interestingly, [15].
restoration of PDX1 protein expression via PDX1 point mutations using
This technique has helped
CRISPR/Cas9 alsostudies
reversedinthese
several areas, such
phenotypes. as for
These gastric
results cancer,
suggest thatwhere
the CRISPR-
mediated correction of point mutations has the potential to improve
in 2015 the study by Gannom and colleagues used the CRISPR/Cas9 technique for the first patient care in the
future [42].
time in GC [44]. This pioneering study evaluated the knockdown effect of dual-specificity
Garcia-Bloj
mitogen-activated protein kinaseet1al. [43], by comparing
(MAP2K1) the ZFN, TALENs,
and the relationship and CRISPR technologies
of MEK-inhibitory drugs for
the overexpression of tumor suppressors in GC strains, demonstrated that CRISPR-dCas9
with cancer cell lines, including gastric cancer. This study was important to evidence the
can restore the expression of the MASPIN/REPRIMO genes, being considered a transient,
RAS/MAPK activation driven by MAP2K1 depletion in gastric cancer.
targeted, and efficient tool for restoring tumor suppressor functions.
CRISPR is an important tool that [9] helps us in a simple and practical way to under-
stand the functions of genes that are still poorly described in GC. A collection of relevant
GC studies that used CRISPR/Cas9 may be found in Table 1.
Genes 2022, 13, 2029 5 of 13

3. Application of CRISPR/CAS9 in Basic GC Research

CRISPR/CAs9 is an extremely versatile tool that can be used in the study and under-
standing of cancer development mechanisms and in the diagnosis and treatment of several
diseases, including GC [15].
This technique has helped studies in several areas, such as for gastric cancer, where in
2015 the study by Gannom and colleagues used the CRISPR/Cas9 technique for the first
time in GC [44]. This pioneering study evaluated the knockdown effect of dual-specificity
mitogen-activated protein kinase 1 (MAP2K1) and the relationship of MEK-inhibitory drugs
with cancer cell lines, including gastric cancer. This study was important to evidence the
RAS/MAPK activation driven by MAP2K1 depletion in gastric cancer.
CRISPR is an important tool that [9] helps us in a simple and practical way to under-
stand the functions of genes that are still poorly described in GC. A collection of relevant
GC studies that used CRISPR/Cas9 may be found in Table 1.

Table 1. Studies performed in gastric cells using the CRISPR/Cas9 methodology.

Functional
CRISPR Gene
Gene Cell Line Phenotype Analyzes Reference
Approach Classification
Performed
Cell proliferation,
KATOIII, SNU16, migration,
FGFR Knockout Oncogene Cell viability [45]
AGS differentiation, and
cell death
Stress-resonant Colony
PDIA3 HFE145 e GES-1 Knockout NE [46]
protein. formation
ATG16L1 AGS, HeLa Knockout Autophagy NE Cell viability [47]
Proliferation,
apoptosis,
colony
PDEF GES, SGC, AGS Knockout Transcription factor Oncogene [48]
formation,
migration, and
invasion
BGC-823, Invasion assay,
SGC-7901 e Cell cycle,
GMAN MKN45, GES-1 Knockout Metastasis. Oncogene proliferation, [49]
HGC-27, and colony
MGC-803, AGS formation
Proliferation,
apoptosis,
colony
PIWIL1 AGP01 Knockout Cell proliferation Oncogene [50]
formation,
migration, and
invasion
TMK-1, AGS,
KATO III,
NCI-N87,
MKN-1, MKN-28,
Proliferation,
MKN-45, SNU-1, Regulation of
apoptosis,
MicroRNA-21 SNU-5, SNU-216, Knockout prostaglandins in Oncogene [51]
colony
SNU- 484, carcinogenesis
formation
SNU-601,
SNU-638,
SNU-668 e
SNU-719
Genes 2022, 13, 2029 6 of 13

Table 1. Cont.

CRISPR Gene Functional

Gene Cell Line Phenotype Analyzes Reference
Approach Classification
Performed
Tumor Cell cycle, cell
MRFAP1 AGS, SGC-7901 Knockout Cell cycle [52]
suppressor viability
Viability, colony
AGS primary Tumor formation,
LMX1A Knockout Transcription factor [53]
cells C-1/GC-2 suppressor TUNEL (cell
death)
Cell adhesion
related to the
CEACAM AGS, KatoIII Knockout NE NE [54]
carcinoembryonic
antigen
Apoptosis,
Proinflammatory
ASC AGS, MKN1 Knockout Oncogene colony [55]
cytokine
formation assay
LNC RNA
promoter of RNA Cell viability,
activated by proliferation,
PANDAR AGS SNU-1 Knockout Oncogene [56]
damage to and colony
antisense DNA formation
CDKN1A
Cell Viability,
LDH Release
SGC-7901, Cell death by Tumor
GSDME Knockout Assay, Cell [57]
MKN-45, HL-60 pyroptosis suppressor
Death, and
Apoptosis
BGC-823, AGS Tumor
REPRIMO Knockout Cell cycle MTT [58]
GES-1 suppressor
Proliferation,
MKN45 Post- migration,
Tumor
miR-30a SGC-7901 Knockout transcriptional colony [59]
suppressor
HEK293T regulation formation,
viability
Proliferation,
BGC823, HGC27, Promotes
colony
MGC803, migration and
SPOCD1 Knockout Oncogene formation, [60]
SGC7901, apoptosis reduction
migration, and
MKN28 GES1 in CG
invasion
Proliferation,
BGC823, HGC27,
colony
MGC803, Adaptive immune
BTN3A2 Knockout Oncogene formation, [60]
SGC7901, response
migration, and
MKN28 GES1
invasion
HEK293T,
AEP MKN45 e Knockout Lysosomal protein Oncogene Cell viability [61]
SGC7901
Cell viability,
HEK293T, MCF7, Mammary protease Tumor
MASPIN Knockin apoptosis, and [43]
SUM159, H157 serine inhibitor suppressor
proliferation
Cell viability,
HEK293T, MCF7, Tumor
REPRIMO Knockin Cell cycle apoptosis, and [43]
SUM159, H157 suppressor
proliferation
Genes 2022, 13, 2029 7 of 13

Table 1. Cont.

CRISPR Gene Functional

Gene Cell Line Phenotype Analyzes Reference
Approach Classification
Performed
Cell viability,
Migration and
EphB2 HGC27 Knockin Oncogene apoptosis, and [62]
invasion
proliferation
Cell viability,
Migration and Tumor
SST 293T and BGC823 Knockout apoptosis, and [63]
invasion suppressor
proliferation
NE: not evaluated.

3.1. Cell Viability and Proliferation

Needless to say, the ability of cells to proliferate rapidly and uncontrollably is a key
factor in GC. Therefore, understanding these biological mechanisms and genes is extremely
important for the identification of therapeutic targets. The CRISPR/Cas9-mediated knock-
out of apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain
(ASC) blocked IL18 and augmented apoptosis in human GC cells, helping to reveal a novel
pro-tumorigenic ASC/IL18 signaling axis in GC cell survival and a candidate therapeutic
target in this disease [55].
Contributing to the findings of mir-21-induced loss of 15-hydroxyprostaglandin de-
hydrogenase (15-PGDH) in early GC and its phenotypic consequences, another study
suppressed 15-PGDH in GC cells using CRISPR/Cas9, which demonstrated increased GC
cell proliferation [51]. Therefore, the study suggested that maintaining the 15-PGDH en-
zyme activity could be a strategy for preventing GC, especially in tubular adenocarcinoma.

3.2. Cell Cycle Control

The study of genes that influence the cell cycle allows us to identify important cellular
processes that differentiate a tumor cell from a healthy cell. Aspects such as unrestrained
growth without the influence of external factors, the loss of programmed cell death capacity,
and also the repression of tumor suppressor genes, among other properties, are important
to identify specific biomarkers and potential therapeutic targets in GC.
To demonstrate the involvement of DNA hypermethylation on the regulation of the
tumor suppressor gene REPRIMO and the TP53-dependent G2 arrest mediator homolog (RPRM)
in GC, Lai and collaborators [58] used the CRISPR/Cas9 technology to first knockdown
DNA methyltransferases (DNMTs). The knockdown of DNMT3A and DNMT3B resulted
in a significant increase in RPRM mRNA by decreasing the RPRM promoter methylation
in GC cells, suggesting an inverse correlation between DNMT functions and RPRM gene
expression. To confirm the tumor suppression role of RPRM, the authors generated RPRM-
deficient GC cell lines using CRISPR/Cas9, which were further inoculated in mice. The
loss of RPRM enhanced the tumor formation in the in vivo model, confirming the role of
RPRM as a tumor suppressor gene in GC. This study provided information regarding the
role of RPRM and its regulatory methylation mechanism in GC development with potential
application as a therapeutic target.
To regain the tumor suppression gene function, a study reactivated the RPRM ex-
pression using CRISPR/dCas9 linked to VP64 and SAM effector domains in GC cell lines,
showing a hypermethylated RPMR promoter and expressing very low basal levels of the
gene [43]. As a result, a marked reduction in GC proliferation was observed. This result
outlined the advantage of this combinatorial epigenome editing approach to reactivate
highly methylated tumor suppressor genes as a promising therapy for GC.
Another promising tumor suppressor was described in the study by Hu and collabo-
rators [52], with the knockout of the Morf4-family-associated protein 1 (MRFAP1) gene using
CRISPR/Cas9 from the CG human cell lines. The researchers observed that the knockout
promoted an interaction of this MLN4924 subtract with suppressed tumor proteins, such
Genes 2022, 13, 2029 8 of 13

as p27, which promoted a decrease in vitality, increased cell cycle arrest, and apoptosis,
data that are important to relate the gene–subtract interaction to favor improvements in
cancer treatment. The study by Liu et al. [56] used the CRISPR/Cas9-mediated knockout
of promoter of CDKN1A antisense DNA damage-activated RNA (PANDAR) and evaluated
its effects on the phenotype of GC cell lines. The knockout of PANDAR suppressed the
proliferative activity and colony formation in the GC cell line. It was also observed by
flow cytometry that the PANDAR knockout blocks the progression of the cell cycle at the
G1/S checkpoint. Through this unique pattern of transcriptional modification, PANDAR
remarkably facilitated the proliferation of cancer cells, the formation of clones, and resis-
tance to chemotherapy. Another study developed by Zhang and colleagues [53] used the
CRISPR/Cas9-mediated knockout of LIM homeobox transcription factor 1 α (LMX1A) in GC
cell lines to identify LMX1A as a primary target of miR-9. The authors that demonstrated
the knockout of LMX1A increased the cell viability and cell proliferation, reinforcing the
role of LMX1A as a tumor suppressor in GC.

3.3. Invasion and Migration

The basic mechanism of metastasis development involves several important charac-
teristics, such as the ability of cells to detach themselves from the primary tumor site and
migrate to colonize more distant sites in the organism [64]. Due to the lack of early diagnosis
and effective therapy options for GC, a better understanding of the mechanisms involved
in the metastatic process of GC is necessary. In this sense, Zhou and colleagues [49] used
the CRISPR/Cas9-mediated knockout of gastric cancer metastasis-associated long non-coding
RNA (GMAN) IncRNA and evaluated the phenotype of GC cells. The knockout of the
GMAN lncRNA delayed the invasive activity of these cells. This study also showed the
overlapping relationship between GMAN and ephrin A1, in which the GMAN knockout
induced an ephrin A1 expression reduction. Assessing the effect of ephrin A1 knockout
in GC cell lines, it reduced the ability for invasion and metastasis in in vivo experiments.
Another important study was developed by Zhu and colleagues [60], in which a GWAS
analysis was used to find low-frequency genetic variants associated with the risk of GC. The
authors found two genes that contained a variant associated with GC risk, SPOC-domain-
containing 1 (SPOCD1) and Butyrophilin subfamily 3 member A2 (BTN3A), and eliminated
these genes using CRISPR/Cas9 in GC cell lines. The knockout of SPOCD1 and BTN3A2
inhibited cell proliferation and colony formation. To investigate the effects of SPOCD1 and
BTN3A2 knockout on migration and invasion, the authors performed xenograft assays and
observed a tumor growth reduction in a rat model associated with SPOCD1 knockout. On
the other hand, BTN3A2 was suggested as a susceptibility gene, although no significant
changes in the xenograft model were observed. Zhang et al. [48] demonstrated that the
CRISPR/Cas9-mediated deletion of SAM-pointed domain-containing Ets transcription factor
(PDEF) inhibited the apoptosis, colony formation, migration, and invasion of GC cell lines.
These results confirm the involvement of PDEF in the different stages of GC development.
The study by Araújo et al. [50] demonstrated that the CRISPR/Cas9-mediated elimination
of the Piwi-like protein 1 (PIWIL1) gene decreased GC cell migration and invasion abilities,
demonstrating the oncogenic role of PIWIL1. On the other hand, Chen Wei’s study [63]
demonstrated that the CRISPR/Ca9-mediated knockout of Somatostatin (SST) significantly
promoted the migration and invasion capabilities of GC cell lines. These data are essential
for characterizing SST as a potential tumor suppressor in GC.
Another interesting study used the CRISPR/Cas9-mediated knockin of ephrin type-
B receptor 2 (EphB2) and evaluated its functions as an independent prognostic marker
in patients with GC [62]. The results indicated that the activation of EphB2 in GC cells
increased the malignant properties of GC cells, reducing the adhesion but accelerating the
migration and invasion capabilities. These results indicate that EphB2 plays a pro-tumor
role in GC and has therapeutic potential to be used in this neoplasia.
Genes 2022, 13, 2029 9 of 13

3.4. Tumorigenesis Models

Helicobacter pylori is a Gram-negative spiral bacterium that is present in 58% of the
global population [65]. Most individuals infected with H. pylori are asymptomatic, and
the presence of this bacterium increases the risk of developing ulcers and gastric adeno-
carcinoma [66]. An interesting study was carried out by Hu and collaborators [49], who
sought to demonstrate the mechanisms in which vitamin D3 can assist in the defense of
the host by promoting an autophagic reaction in the fight against H. pylori. The results
showed that there is a new pathogenic mechanism that H. pylori can survive by hiding
inside the autophagosomes in the GC cells by using the CRISPR/Cas9-mediated knockout
of protein disulfide-isomerase A3 (PDIA3). From this study, a new vitamin D3 signaling
pathway activates the PDIA3-STAT3-MCOLN3-Ca 2+ axis to reactivate the lysosome.
The molecular mechanism by which H. pylori induces peptic ulcers or gastritis cancer
is not understood, but it probably involves a combination of host genetic predisposition and
bacterial virulence factors (e.g., VacA and CagA proteins) [65]. The vacuolating cytotoxin
(VacA) is responsible for several cellular responses, such as cell vacuolization, as well as
other processes such as autophagy and necrosis [67]. Foegeding and collaborators [47]
inhibited autophagy by using the CRISPR/Cas9-mediated knockout of autophagy-related
16-like 1 (ATG16L1) in HeLa cells. The results showed increased VacA levels or increased
vacuolization compared with the control and that the VacA degradation is independent of
the autophagic activity. CagA is a virulence factor used for the detection of H. pylori and is
considered an important risk factor for severe gastric diseases, including GC. Zhao and
coauthors [54] evaluated the effects of integrin receptors and the function of cell adhesion
molecule 1 receptors (CEACAM) through the cag-type IV secretion system (cag-T4SS) on
the CagA translocation process through a multiple knockout of CEACAM receptors in the
GC cell line. The results showed that neither the direct interaction of the components of
cag-T4SS with the integrins nor any signaling event mediated by the integrin is necessary
for the translocation of CagA. Furthermore, the CRISPR/Cas9 mediated the deletion of
miR-30a in H. pylori infected mice, the knockout mice demonstrated that genetic editing did
not affect the growth and development of the mice, and little effect was observed on the H.
pylori colonization rates of the mice. Increased incidence rates of chronic gastritis, chronic
atrophic gastritis, atypical hyperplasia, and other precancerous lesions and manifestations
of adenocarcinoma in the antral or gastric mucosa of rats have also been reported. These
data demonstrate that miR-30a plays the role of a tumor suppressor in GC [59].

3.5. Chemotherapy Response

From the development of tumor cells to create resistance to chemotherapeutic-induced
cell death, the CRISPR tool has been used to discover new therapeutic targets and drugs for
the treatment of GC. Wang and colleagues [57] used CRISPR/Cas9 to mediate the knockout
of the gasdermin E (GSDME) gene in a GC cell line to assess the effect of 5-fluoracil on the in-
duction of pyroptosis in these cells. The authors found that the GSDME deficiency changed
the pyroptosis induced by 5-FU to apoptosis, characterized by shrinkage, fragmentation in
apoptotic bodies, and cell death without lysis. The study by Cui and collaborators [61] used
the CRISPR/Cas9-mediated knockout of the legumain (AEP) gene to assess the proliferative
capacity of these cells in the presence of different chemotherapeutic agents. This gene has
previously been shown to be an oncogene related to invasiveness and metastasis in GC. The
authors demonstrated that AEP knockout GC cells caused significantly decreased prolifera-
tion after treatment with 5-FU, paclitaxel, docetaxel, and T-DM1. These data demonstrate
that AEP is a potential therapeutic target for GC.

4. Application of CRISPR/CAS9 in GC Clinical Research

Due to the ease of use that CRISPR/Cas9 has brought to gene editing, the chances of
treating diseases previously defined as “incurable” has greatly increased. However, this
technology is a challenge in the field of clinical studies related to GC, due to the difficulties
Genes 2022, 13, 2029 10 of 13

in finding a delivery system that is specific enough and capable of targeting only the cells
of interest inside a given tissue.
There are currently two clinical trials where CRISPR/Cas9 is being used for the treat-
ment of patients with GC via different approaches. The “Phase I/II Trial in Patients with
Metastatic Gastrointestinal Epithelial Cancer Administering Tumor-Infiltrating Lympho-
cytes in which the Gene Encoding CISH was Inactivated using the CRISPR/Cas9 System”
(NCT04426669) is one of the pioneers in this type of study, which is a clinical trial to evalu-
ate the safety and efficacy of genetically modified neoantigen-specific tumor-infiltrating
lymphocytes (TILs), in which the intracellular immune checkpoint CISH (cytokine-induced
SH2 protein) will be inhibited using CRISPR/Cas9 gene editing for the treatment of gas-
trointestinal (GI) cancer [68].
Another interesting ongoing clinical trial is “PD-1 Knockout EBV-CTLs for Advanced-
Stage Epstein–Barr Virus (EBV)-Associated Malignancies”. This clinical trial seeks to collect
peripheral blood lymphocytes from patients with advanced stage Epstein–Barr virus (EBV)
GC to generate PD-1 (programmed cell death protein 1) knockout cytotoxic T-lymphocytes
(CTL). After the reintroduction of PD-1 knockout EBV-CTL, the patients are expected to
show increased GC progression-free survival (PFS) and overall survival (OS) rates [69].
These studies are important for understanding the administration of CRISPR in
humans and establishing this technique as a future personalized treatment in patients
with GC.

5. Conclusions and Future Perspectives

Over the years, CRISPR technology has given us new perspectives that were previously
unattainable in the scientific world. In the context of GC, CRISPR has been an ally in the
discovery of new mechanisms and genes that play key roles in this neoplasm. In addition
to helping to understand the molecular mechanisms involved in the emergence of GC,
CRISPR is a promising tool with the potential to modify specific genes important for
gastric carcinogenesis and reverse processes as we have never been able to before. CRISPR
technology has proved to be such an advantageous tool, as we can see its potential via
in vitro, in vivo, and clinical studies, proving it to be a technology that is applicable for
several approaches, including in cancer, but we still have not identified the complete
domain of CRISPR, so there is still a lot of work to be done to refine this technology and
make it applicable.

Author Contributions: Conception and design, R.S.A., F.W. and M.A.C.S.; writing, review and
revision, R.S.A., F.W., B.T.R.K. and M.A.C.S. All authors have read and agreed to the published
version of the manuscript.
Funding: This study was funded by the São Paulo Research Foundation (FAPESP) under grant
numbers 2016/25562-0 and 2019/20592-6; the National Council for Scientific and Technological
Development (CNPq) under grant number 2018/301127-2; and the Coordination for the Improve-
ment of Higher Education Personnel (CAPES) under grant numbers 23038051640/2009-01 and
88882.430350/2019-01. M.A.C.S. was funded by FAPESP grant number 2016/25562-0; CNPq grant
number 2018/301127-2; and CAPES 23038051640/2009-01. B.T.R.K. was funded by FAPESP grant
number 2019/20592-6. R.S.A. was funded by CAPES Doctoral Fellowship 88882.430350/2019-01.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Figures were created with biorender.com.
Conflicts of Interest: The authors declare no conflict of interest.
Genes 2022, 13, 2029 11 of 13

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