World Journal of Microbiology and Biotechnology (2023) 39:58

https://doi.org/10.1007/s11274-022-03504-0

RESEARCH

Targeted gene inactivation in Salmonella Typhi by CRISPR/

Cas9‑assisted homologous recombination
Yousof Tarverdizadeh1 · Mohammad Khalili1 · Saber Esmaeili2 · Gholamreza Ahmadian3 · Mehdi Golchin1 ·
Abbas Hajizade4

Received: 9 September 2022 / Accepted: 19 December 2022 / Published online: 27 December 2022
© The Author(s), under exclusive licence to Springer Nature B.V. 2022

Abstract
Background  Targeted gene inactivation (TGI) is a widely used technique for the study of genes’ functions. There are many
different methods for TGI, however, most of them are so complicated and time-consuming. New promising genetic engineer-
ing tools are developing for this purpose. In the present study, for the first time we disrupted a virulence gene from Salmonella
enterica serovar Typhi (S. Typhi), located in the bacterial chromosome using CRISPR/Cas9 system and homology directed
repair (HDR).
Methods  For this aim, pCas9 plasmid containing Cas9 enzyme and required proteins for homology directed recombina-
tion was transferred to S. Typhi by electroporation. On the other hand, a specific guide RNA (gRNA) was designed using
CRISPOR online tool. Synthetic gRNA was cloned into pTargetF plasmid. Also, a DNA fragment (HDR fragment) was
designed to incorporate into the bacterial chromosome following the cleavage of the bacterial genome by Cas9 enzyme.
pTargetF containing gRNA and HDR fragment were co-transferred to S. Typhi containing pcas9 plasmid. The transformed
bacteria were screened for recombination using PCR, restriction digestion and sequencing.
Results  The results of PCR, restriction digestion and sequencing showed the successful recombination of S. Typhi, in which
the gidA gene is disrupted.
Conclusion  In the present study we aimed to develop a rapid and robust method for targeted gene inactivation in a bacte-
rial species, S. Typhi. This procedure can be exploited for disruption of other Salmonella as well as other bacteria’s genes.

Keywords  Targeted gene inactivation (TGI) · CRISPR/Cas system · Salmonella Typhi, gidA gene · Live attenuated
vaccines, homologous recombination

Introduction

Targeted gene inactivation (TGI) is a proper technique for

* Abbas Hajizade
abbashajizade@gmail.com; abbashajizade@ihu.ac.ir
1
Department of Pathobiology, Faculty of Veterinary
Medicine, Shahid Bahonar University of Kerman, Kerman,
Iran
2
Department of Epidemiology and Biostatics, Research Centre
for Emerging and Reemerging Infectious Diseases, Pasteur
Institute of Iran, Tehran, Iran
3
Department of Industrial and Environmental Biotechnology,
National Institute for Genetic Engineering and Biotechnology
(NIGEB), Tehran, Iran
4
Biology Research Center, Faculty of Basic Sciences, Imam
Hossein University, Tehran, Iran
the study of a gene: its function, its role in a cell’s life, its
association with specific traits, its ability to cause disease,
etc. (Meng et al. 2008; Tolonen et al. 2009; Zhang et al.
2019; Wang et al. 2022). Also, TGI is a major goal in the
treatment of specific diseases like cancer (Setton et al. 2021;
Duffy 2020).
There are many techniques for targeted gene inactivation.
RNA silencing, for example, is a powerful technique, which
has been widely used for this aim (Han and Han 2018; Lev-
anova et al. 2018). In this technique, different oligomers,
including peptide nucleic acid (PNA), oligodeoxynucleo-
tide (ODN), external guide sequence (EGS), locked nucleic
acid (LNA), etc., are used to bind to a specific RNA and
inhibit its translation (Halloy et al. 2022; Singh et al. 2020).

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Page 2 of 9 World Journal of Microbiology and Biotechnology (2023) 39:58

Although it is a specific and efficient method, however, it
provides a temporary inactivation of a gene.
The CRISPR/cas system, a part of bacterial adaptive
immunity, recognizes and degrades exogenous nucleic
acids, such as plasmids and phages (Horvath et al. 2010;
Katalani 2020). Robust and specific recognition of a desired
sequence have made CRISPR/Cas a unique tool for genome
modification (Anzalone et al. 2020). In comparison to other
endonuclease-based genome editing tools, such as transcrip-
tion Activator- like Effector Nuclease (TALEN) and zinc
finger nuclease (ZFNs), this system has many advantages.
For example, TALENs and ZFNs are man-made artificial
protein-based tools, while, CRISPR is derived from bacteria
and is RNA- based. The CRISPR’s efficiency and specificity
is higher than TALENs and ZFNs, but, at the same time, its
cost is lower (Gupta and Musunuru 2014; Kim et al. 2014).
Since its introducing in 2012 for genome editing,
CRISPR/Cas system has been exploited for genome manip-
ulation in both eukaryotes and prokaryotes, especially in
eukaryotes (Feng et al. 2013; Chen et al. 2019; Platt et al.
2014; Barrangou et al. 2016). However, the use of the power
and advantages of this system in bacterial world is still in
the beginning.
Salmonella entericaserovar Typhi (S. Typhi) is the aetio-
logic agent of typhoid fever. Causing nearly 10.9 million
illnesses and 116.8 thousand deaths globally, this bacterium
is a major health problem particularly in developing coun-
tries (Wierzba and Sanders 2019). Investigating the role of
genes coding virulence factors is an essential step for the
development of new drugs and vaccines against the infec-
tious agents. The pathogenicity ofS. Typhi is controlled by
different genes mainly located on Salmonella pathogenic-
ity island-1 (SPI-1) and Salmonellapathogenicity island-2
(SPI-2) (Lee et al. 2000; Cirillo et al. 1998; Lostroh and
Lee 2001). Glucose-inhibited divison (GidA) protein is a
tRNA modification enzyme (Shi et al. 2009) which is present
in different bacteria,Escherichia coli (E. coli), Salmonella
Typhimurium, Streptococcus pyogenes, Pseudomonas aer-
uginosa, Aeromonas hydrophila, etc. It is involved in many
bacterial functions, such as motility, bacterial shape, patho-
genicity, etc. (Shippy 2011).
In the present study, we exploited the CRISPR/ Cas9 sys-
tem to disrupt gidA gene by the insertion of a 39 bp DNA
fragment into the bacterial genome.
Materials and methods
Bacterial strains, plasmids and media
Table 1 represents the bacterial strains and plasmids used in
the present study. A clinically isolated Salmonella enterica
serovar Typhi (S. Typhi) was used for gene inactivation.
Luria Bertani (LB) broth and agar were used for bacterial
growth. When appropriate, 50 µg/mL of kanamycin and/or
100  µg/mL of spectinomycin (Sigma Ltd, Germany) were
added. pCas9 and pTargetF plasmids were prepared from
addgene under the numbers 62,225 and 62,226, respectively.
All media required for biochemical analysis tests were pur-
chased from Gibco (USA).
Generation of salmonella Typhi transformant
carrying pCas9 (STcas9)
Strain confirmation
Biochemical identification tests as well as molecular test
were used for the confirmation of S. Typhi isolate. Indeed,
the pathogenicity of the isolate was approved by bacterial
injection to mice.
IMVIC (Indole, Methyl Red, Voges-Proskauer, and Cit-
rate), TSI (Triple Sugar Iron), SIM (Sulfur, Indole, Motil-
ity) and Urease tests were exploited for biochemical iden-
tification of the strain. For molecular identification of the

Table 1  Strains and plasmids Strain or plasmid Main characteristics Source or reference

used in this study
Salmonella Typhi Hospital isolate Isolated from an in-patient
Genotype: gidA+
LD50 in mice: 7.8 × ­108 ­CFUa
pCas9 Bacterial Resistance: K ­ ANb Addgene (62,225)
Growth Temperature: 30 °C
Growth Strain: DH5alpha
Growth instructions: Must be grown at 30 C! Vector
has temperature-sensitive replication (RepA101ts)
Copy number: Low copy
pTargetF Bacterial resistance: ­SPTc Addgene (62,226)
Growth temperature: 37 °C
Growth strain: DH5alpha
Copy number: High copy
a
 CFU: Colony Forming Unit; bKanamycin cSpectinomycin

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bacterium, two different S. Typhi genes were analyzed by
polymerase chain reaction (PCR): 16 S rRNA and gidA. The
sequences of the primers are listed in Table 2.
Preparation of competent S. Typhi cells
S. Typhi cells were competent for transformation using
chemical method as described by McLachlan et al. (MacLa-
chlan et al. 1985) with a little modification. Briefly, S. Typhi
cells were grown in 5 mL of LB broth till the O ­ D640 of the
culture reached 0.75. Then, cells were collected by centrifu-
gation at 4000×G for 10 min at 4 °C. The Pellet was resus-
pended in 1volume (5 mL) of cold 1mM HEPES buffer and
centrifuged again in the same conditions. The pellet was
then resuspended in 1/2 volume of HEPES buffer, and recen-
trifuged. The final pellet (competent cells) were resuspended
in 1 mM HEPES buffer containing 10% glycerol.
Electro Transformation
pCas9 plasmid was transferred to the competent cells were
via electroporation (MacLachlan et al. 1985). For this aim, 2
µL (900 ng) of pcas9 plasmid was added to 40 µL of chilled
competent cells. The content was transferred to a chilled
electroporation cuvette (Bio Rad). Then, a pulse (200 Ω,
25 µF and 12.5 kv/cm) was applied for 5 ms. Then, 1mL of
chilled SOC broth was added to the cuvette and the content
was transferred to a sterile 1.5 mL microtube and incubated
in 30 °C for 3 h with gentle shaking. After 3 h of incubation,
cells were cultured on a LB agar plate containing 50 µg/mL
of kanamycin.
Confirmation of the bacterial transformation:
After 36 h incubation in 30 °C, grown colonies were ana-
lyzed for transformation using PCR. For this aim specific
primers for pCas9 plasmid were used. The sequence of the
primers was as follows:
Forward primer: 5′CAT​AAT​TCG​TGT​CGC​TCA​AG3′;
Reverse primer: 5′ACG​AAG​AAT​CCA​TGG​GTA​TG3′.
Resulted PCR products with these primers will have
lengths of 297 bp.
Table 2  The sequence of the Gene᾽s name
primers for the amplification of
16s rRNA and gidA genes 16 S rRNA
gidA
sgRNA design and construction of sgRNA plasmid
sgRNA was designed using CRISPOR tool (crispor.tefor.net)
to identify crRNA spacer sequences with the highest speci-
ficity score (> 50). The sequence was sent for the synthesis
to SinaClone Company (Karaj, Iran). Then, this sequence
was cloned into pTargetF plasmid (from addgene under the
number 62,226) and the transformed clones were confirmed
by PCR and sequencing.
Construction of HDR oligo fragment
An HDR (homology-directed repair) fragment designed and
constructed to direct the desired sequence to the specific
site in the bacterial genome. The HDR fragment was com-
posed of three regions: an upstream arm (50 bp), an inser-
tion segment (39 bp) and a downstream arm (49 bp). The
sequences of upstream and downstream arms were the same
as the sequence of upstream and downstream of the PAM
site, respectively. The insertion segment (39 bp) composed
of a short segment of the beginning of gidA gene (28 bp)
and a stop codon. The stop codon was applied to the inser-
tion sequence in order to inhibit the translation of the entire
length of gidA mRNA.
Transfer of sgRNA plasmid and HDR fragment
to STcas9
Following the construction of sgRNA plasmid and HDR
fragment, they were transferred to STCas9 using heat- shock
method (Ebrahimi 2018). Briefly, 3 µL of sgRNA plasmid
50 mg/µL) and 5 µL of HDR fragment (200 mg/µL) was
added to competent cells (HDR provides the upstream
and downstream homology arms as well as the insertion
sequence). Stcas9 cells were competent bFIGy chemical
method using C ­ aCl2. The samples were placed on ice for
0.5 h. Then, the samples were put in a water bath (37 °C)
for 90 S and then placed on ice for 5 min. After that, 1 mL
of LB broth medium was added to each tube and tubes were
incubated at 37 °C for 3 h. The cells were collected using
centrifugation at 8000×G for 2 min and cultured on LB Agar
plates containing 50 µg/mL kanamycin and 50 µg/mL spec-
tinomycin. The plates were incubated at 30 °C for 24 h and
grown colonies were tested for recombination.
Primer sequence Amplicon size
Forward: CCG​GGA​ACT​CAA​AGG​AGA​CT 174 bp
Reverse: ACT​CCA​ATC​CGG​ACT​ACG​AC
Forward: GCG​GTC​TGA​TGG​CGA​AAG​CGATC​ 453 bp
Reverse: TTG​AGC​CAA​CAC​GCT​GAA​ATC​GAT​AG
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Analysis of the bacterial genome modification Results

To investigate if recombination has been done, three strat- Generation of STcas9

egies were employed: PCR, restriction digestion, and
sequencing. Strain confirmation
PCR.
To confirm the recombination via PCR, a pair of primers The results of biochemical identification test are presented
were used. The sequence of the primers was as follows: in Table 3. As can be seen, the investigated strain had the
F primer: ATG​TTT​TAT​CAG​GAT​CCT​TTT​GAC​GTCA; typical properties of S. Typhi.
R primer: GTT​GAG​CCA​ACA​CGC​TGA​AAT​CGA​TAG. Figure 1 shows the results of the amplification of rRNA
As noted, the insertion fragment contained the F primer; and gidA genes. The results confirmed that the strain in
so, in recombinant samples we will have 2 bands: a 680 bp gidA+ S. Typhi.
band related to the sequence of the beginning of the gene;
and a 601 bp band related to the inserted sequence.
Restriction Digestion.
Since there is the SalI endonuclease restriction site in the
insertion fragment, following the PCR with the above prim-
ers, the upper band will be digested by SalI.
Sequencing.
Finally, the samples that were PCR positive sent for
Sanger sequencing with the reverse primer (GTT​GAG​CCA​
ACA​CGC​TGA​AAT​CGA​TAG).

Characterization of the recombinant ΔgidA S. Typhi

Investigating the growth of ΔgidA S. Typhi

To investigate the effect of the disruption of gidA gene on

the growth rate of the bacterium, wild-type and recombinant
strains were cultured in LB broth media at 37 °C with aera-
tion at 150 rpm. The cultures continued until the bacterial
cultures reached the stationary phase.

Investigating the  pathogenicity of  ΔgidA S. Typhi The

effect of the disruption of gidA gene on the pathogenicity
of S. Typhi was investigated by determining the ­LD50 of the
mutant and wild type S. Typhi bacteria in mice. For this
aim eight-week-old BALB/c mice were divided into five
groups of five animals each. Animals in groups 1, 2, 3, and
4 received 1­ 06, ­107, ­108, and 1­ 09 CFU of bacteria peritone-
ally, respectively. Mice in group 5 received PBS. Mice were
monitored for 5 days and the mortality among groups was
recorded. The ­LD50was determined using Behrens and Kar- Fig. 1  Results of the amplification of rRNA and gidA genes. Lanes 1
ber method (Behrens and Karber 1983). and 2: Amplification with 16S rRNA specific primers. Lanes 3 and 4:
Amplification with gidA specific primers. Lane 5: Control. Lane 6: 1
kb DNA ladder

Table 3  The results of TSI MR VP H2S Urease Motility Indol CO2 Citrate

biochemical identification test
K/A + − + − + − − −

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Confirmation of the bacterial transformation
Following the preparation of S. Typhi competent cells, and
the transfer of pCas9 plasmid to these cells, the transfor-
mation of the cells was investigated using PCR. Figure 2A
shows the bacterial growth on LB agar plate containing
50 µg kanamycin. The grown colonies were analyzed for
the transformation using colony-PCR with specific primers
that amplify a 453 bp segment of gidA gene and a 297 bp
fragment of pCas 9 plasmid. As can be seen in Fig. 2B, a
297 bp band has been amplified in 3 analyzed colonies, that
confirms the entrance of pCas9 plasmid to S. Typhi cells.
Fig. 2  A Grown colonies on theLB agar plate containing 50 μg kana-
mycin following the electro-transformation.B  Investigation of the
entrance of pCas9 plasmid to S. Typhi competentcells. The PCR was
carried out by two sets of primers: specific primers thatamplifies a
453 bp segment of gidA gene and specific primers for a 297bp frag-
ment of pCas 9 plasmid. Lanes 1, 2, 4 and 5: PCR results of 4 colo-
niesgrown on agar medium after transformation. Lane 6 is negative
control. Lane 7Wild strain S. Typhi. Lane 3: 1 kb DNA ladder.
Construction of sgRNA plasmid
Designed gRNA had the following sequence:
Watson strand: 5′-CGC​GAT​GGC​CGC​AGC​GCG​TA-3′;
Crick strand: 5′-TAC​GCG​CTG​CGG​CCA​TCG​CG-3′.
The designed gRNA was synthesized chemically (Sina-
Clone, Iran) and cloned into pTargetF plasmid. The recom-
binant plasmid was transferred to E. coli DH5α competent
cells via heat shock method. Following the growth of the
colonies on LB agar medium, transformed colonies were
confirmed via PCR and sequencing. Figure 3A shows the
result of the PCR. Three sets of PCR reactions were carried
out: (1) Internal forward primer of pTargetF plasmid + Crick
strand of gRNA as the reverse primer; (2) Watson strand
as the forward primer and an internal primer of pTargetF
plasmid as the reverse primer; (3) Internal forward primer
of pTargetF plasmid + an internal primer of pTargetF plas-
mid as the reverse primer. As can be seen in Fig. 3A all
three bands were observed following the PCR. The result
of sequencing that can be seen in Fig. 3B further confirmed
that the gRNA has been successfully inserted to pTargetF
plasmid.
Constraction of HDR oligo fragment
The final sequence of HDR oligo fragment is shown in
Fig. 4A. it is a 139 bp fragment comprised of an upstream
arm (50 bp), an insertion fragment (39 bp), and a down-
stream arm (49 bp). Figure 4B represents the HDR oligo
fragment in a 2% agarose gel.
Analysis of the bacterial genome modification
Confirmation of the insertion of the desired sequence (a
39 bp fragment) to the chromosome of S. Typhi was per-
formed using PCR, restriction digestion and sequencing.
Figure 5A Shows the result of PCR amplification of two
segments of the chromosomes. As stated before, a part of
the insert sequence (39 bp fragment) is the initial part of
gidA gene. Therefore, if the insertion occurs, following the
PCR using This fragment as forward primer, two fragments
should be seen: a 601 bp fragment and a 680 bp fragment.
As can be seen in Fig. 5A, two fragments are observed that
shows the insertion has occurred successfully. Indeed, the
amplified fragment were undergone restriction digestion
with SalI restriction enzyme. Since the restriction site of
this enzyme is present in the insert sequence, digestion with
this enzyme leads to the disappearing of the upper band. Fig-
ure 5B shows that following restriction digestion the upper
band has been disappeared; therefore, it confirms the inser-
tion of the fragment to the bacterial chromosome. Finally,
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Page 6 of 9 World Journal of Microbiology and Biotechnology (2023) 39:58

Fig. 3  A Investigation of theen-

trance of gRNA to pTargetF
plasmid. The PCR was carried
out by three sets ofprimers:
1. Internal forward primer
of pTargetF plasmid + Crick
strand of gRNAas the reverse
primer (lane 1); 2. Watson
strand as the forward primer and
aninternal primer of pTargetF
plasmid as the reverse primer
(lane 2); 3. Internalforward
primer of pTargetF plasmid +
an internal primer of pTargetF
plasmid asthe reverse primer
(lane3). Lane 3: 1 kb DNA
ladder. B Sequencing result.
Ascan be seen, Watson strand of
the gRNA has been sequenced
exactly, which meansthat the
gRNA has been cloned success-
fully

Fig. 4  A The sequence of the-

HDR fragment. From the left:
TG was added in order to the
stop codon (TGA) be locatedin
the frame. The sequence GTC​
GAC​is SalI endonuclease
restriction site. Thelast part of
the sequence (green letters) is
the sequence of the initial partof
gidA gene. The sequence was
placed in the insert fragment
for the easyconfirmation of the
insertion of the fragment into
the bacterial genome. B HDR-
fragment in a 2% agarose gel.
Lanes 1 and 2: HDR fragments;
Lane 3: DNA Ladder

sequencing showed that the insert sequence has successfully
inserted into the bacterial genome in the precise location
(Fig. 5C).
Characterization of the recombinant ΔgidA S. Typhi
Investigating the in vivo pathogenicity of ΔgidA S. Typhi
LD50 of the recombinant and wild-type S. Typhi was cal-
culated as 7.8 × ­108 and 5 × ­107, respectively, which shows
about 15 times reduction in the pathogenicity of the recom-
binant strain.

Investigating the effect of the disruption of gidA

on the growth of S. Typhi Discussion

The bacterial growth curves of the recombinant and wild- In the present study, for the first time, we disrupt a Salmo-
type S. Typhi is presented in Fig. 6. As can be seen in the nella Typhi gene via CRISPR-aided homologous recombina-
figure, gidA knockdown resulted in the reduction of the bac- tion. A 39 bp fragment was inserted into gidA gene, located
terial growth. on the bacterial chromosome.

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World Journal of Microbiology and Biotechnology (2023) 39:58 Page 7 of 9  58
Fig. 5  Confirmation of the recombination.A PCR amplification of a
fragment containing the insert sequence (the 39 bpfragment). From
seven analyzed colonies only one colony (Clone 2, Lane 2) was-
successfully recombinant. Lane 8: 1 kb DNA ladder. B Restriction
digestion ofthe amplified fragment by SalI enzymr. Lane 1: Con-
trol (without SalI enzyme);Lane 2: Test. Lane 3: 1 kb DNA ladder.
gRNA was designed so that the Cas9 enzyme cut the
initial part of the gene. Since pCas9 plasmid carries the
required genes for homologous recombination, following
the cleavage with cas9, the desired fragment will be inserted
into the cleaved region.
Using PCR, we showed that S. Typhi can maintain exog-
enous plasmids for multiple rounds of subcultures of the
bacteria.
C Sequencing result. Sequencing wasperformed using the reverse
primer. As can be seen, the 39 bp fragment has beeninserted into
the bacterial genome. Note: Since the sequencing was done usingthe
reverse primer, the highlighted sequence is the reverse complement of
theinserted fragment
gRNA was designed so that the Cas9 endonuclease
cleave the initial part of the gene (nucleotides 79 and 80
of the coding sequence) to ensure the inactivation of the
gidA gene᾽s product, GidA protein. Via this strategy the
recombinant bacteria can be used in different studies,
including use as a live-attenuated vaccine candidate, inves-
tigation of the effect of the desired gene on the survival
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Page 8 of 9
Fig. 6  The growth curves ofwild-type (WT) and ΔgidA mutant S.
Typhi. The growth rate of themutant strain is obviously slower than
the wild-type
and pathogenicity of the bacteria, the effect of gene on the
morphology of the bacteria, etc.
As it was stated before, it is the first report on the use
of CRISPR for disruption of a gene in Salmonella; how-
ever, there are some studies that have used this system to
knockout genes in other bacteria. Zhang et al. used CRISPR/
Cas system to knocked out an antibiotic- related gene, oqxB
in Pseudomonas fulva, a Gram-negative bacterium (Zhang
et al. 2022). Like the present study, they used a two-plas-
mid strategy for this aim. They exploited pCasPA plas-
mid (17,653 bp) to express Cas9 enzyme and pACRISPR
(6936 bP) for the expression of sgRNA. They observed a
much higher recombination efficiency than of our study. It
may be due to the use of electroporation for bacterial trans-
formation (in our study pTargetF plasmid and HDR fragment
were transferred to the STCas9 using heat-shock method).
Indeed, they cloned the oligo arm in pACRISPR and it can
be another reason for higher recombination rate in their
experiment.
Wijaga et  al., used a similar two- plasmid system to
manipulate Clostridium beijerinckii᾽s genome (Diallo
et al. 2020). They employed pE_X_cas9 (12,849 bp) and
­pS_XRKR (6336bP) for the expression of Cas9 enzyme and
gRNA, respectively. They deleted a 2379 bp DNA fragment
from a chromosomal gene (spoIIE) that is involved in sporu-
lation and inserted a 1680 bp gene (N. patriciarum celA
gene) into the chromosome.
Hou et al. exploited a two-plasmid system for deleting a
genetic locus containing eltIIc1, estA, estB, eltI, and faeG in
enterotoxigenic E. coli(ETEC). They improved the recom-
bination efficiency by increasing the homology arm (from
50 bp to 300 bp) (Hou et al. 2020).
One limitation of the present study was the low recombi-
nation rate. We tested about 50 colonies grown on LB agar
containing kanamycin and spectinomycin. Only one colony
out of 50 colonies was recombined in the desired locus.
Maybe this low rate of the recombination was related to
our strategy for the transfer of oligo fragment and pTargetF
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World Journal of Microbiology and Biotechnology (2023) 39:58
plasmid. We used heat-shock method to transform the bac-
teria. Since oligo fragments are not replicated in the bacte-
rial cell and are destroyed immediately, in this method we
observed a low rate of recombination. Using electropora-
tion may increase the transfer of oligo fragment as well as
plasmids into the bacteria and as a result it improves the
recombination rate. Another possible reason for the low
recombination rate can be related the size of the homology
arms. In our study we used 50 and 49 bp sequence for the
upstream and downstream arms, respectively. In the study
by Hou, they showed that this size is so small for an efficient
recombination (Hou et al. 2020).
Investigation and comparison of the growth rate of the
mutant ∆gidA and wild-type S. Typhi showed that ∆gidA
strain has a much slower rate than the wild-type strain. This
shows the role of GidA in the regulation of S. Typhi path-
ogenicity. These results are compatible with the previous
studies on S. Typhimurium (Rehl et al. 2013), Streptococ-
cus suis (Gao et al. 2016), and E. coli(von Meyenburg et al.
1982).
Following the disruption of gidAgene, the in vivo patho-
genicity of the mutant strain decreased significantly. These
findings are in agreement with different studies, including
Shippy et al., (in S. Typhimurium) (Shippy 2011), Sha et al.,
(inAeromonas hydrophila) (Sha et al. 2004), and Kinscherf
and Willis (in Pseudomonas syringae) (Kinscherf and Willis
2002). Reduction in the pathogenicity of ∆gidA strain con-
firms the role of GidA protein in the regulation of S. Typhi
virulence factors. However, there should be more research
to find the affected virulence factors.
All and all, the result of this study revealed the potential
(applicability) of CRISPR/cas9 system for genome editing
of Salmonella Typhi. It can pave way for the study of the
function of the bacterium’s genes, Indeed, it is possible to
develop new live-attenuated vaccine candidates and other
industrial strains.
Acknowledgements  This study is a part of Y.T. PhD thesis. Authors
thanks the staff of pathobiology Department of Shahid Bahonar Uni-
versity of Kerman and Biology Research Center of Imam Hossein
University.
Declarations 
Conflict of interest  The authors have no relevant financial or non-fi-
nancial interests to disclose.
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