Coupling CRISPR/Cas9 and Lambda Red Recombineering System for Genome Editing of Salmonella Gallinarum and the Effect of ssaU Knock-Out Mutant on the Virulence of Bacteria

The poultry industry in developing countries still faces a significant threat from fowl typhoid, a disease caused by Salmonella Gallinarum that has been well contained in more economically developed countries. In addition to the virulence exhibited by large virulence plasmid (85 kb), Salmonella Pathogenicity Island 2 in S. Gallinarum plays a key role in mediating disease through its type III secretion systems (TTSS). The TTSS secrete effector protein across the Salmonella containing vacuoles and mediate the internalization of bacteria by modulating vesicular passage. In this study, candidate virulent ssaU gene (~1 kb) encoding type III secretion system was successfully deleted from indigenously isolated S. Gallinarum genome through homology-directed repair using CRISPR/Cas9 and lambda recombination systems. CRISPR/Cas9-based genome editing of poultry-derived Salmonella Gallinarum has not been previously reported, which might be linked to a lack of efficiency in its genetic tools. This is the first study which demonstrates a complete CRISPR/Cas9-based gene deletion from this bacterial genome. More importantly, a poultry experimental model was employed to assess the virulence potential of this mutant strain (ΔssaU_SG18) which was unable to produce any mortality in the experimentally challenged birds as compared to the wild type strain. No effect on weight gain was observed whereas bacteria were unable to colonize the intestine and liver in our challenge model. This in vivo loss of virulence in mutant strain provides an excellent functionality of this system to be useful in live vaccine development against this resistant and patho genic bacteria.


Introduction
Salmonella enterica sub sp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) is a crucial pathogen of poultry causing fowl typhoid (FT), an acute septicemic disease leading to catarrhal enteritis, hepatitis, pericarditis and splenomegaly [1]. With a mortality rate of 40-90%, the disease causes havoc in terms of decreased weight gain in broilers and low egg production in layer birds [2]. This disease spreads through both horizontal and vertical transmission [3]. Currently, live attenuated strain (9R) with a semi-rough lipo-polysaccharide structure has been used as vaccine to control FT in poultry [4,5]. In the last two decades, various outbreaks of FT have been reported in India, Nigeria, Korea and Japan [4][5][6][7]. In addition, the emerging resistance of S. Gallinarum against ciprofloxacin, amoxicillin, erythromycin, tetracycline and doxycycline reported in recent studies demonstrates an alarming situation in the future for poultry industry [8]. This urges the undermining of the virulence factors and molecular pathways that contribute to SG's pathogenicity. The enormous 85 kb plasmid of Salmonella Gallinarum is crucial for virulence, and various plasmid-borne virulence genes have been characterized for their virulence potential [9]. Salmonella pathogenicity island 2 (SPI-2) has been shown to play a crucial role in disease transmission by Salmonella enterica via type III secretion system apparatus (TTSS) [10].
The SPI-2 encoded TTSS gets activated and interfere with the maturation of phagosome once the Salmonella is engulfed. This results in the formation of specified Salmonella Containing vacuole (SCV), leading to intracellular survival and replication of Salmonella [11].
A number of steps precede the SCV formation, including endocytic pathway hence inhibiting the delivery of hydrolytic enzymes; relocation of SCV to perinuclear area and development of Salmonella-induced-filaments (Garcia-del Portillo et al., 1993). Due to bacterial intervention with many cellular processes these events proceeds to the survival, colonization, and spread of Salmonella. Hence SPI-2-encoded TTSS is crucial to orchestrate SCV formation and intracellular survival of Salmonella [12].
The genes belonging to SPI-2 including ssaG, ssaV, SsaU and ssrA have been classified as potential virulent factors for Salmonella infections [13]. These TTSS-associated genes regulate the trafficking of several virulence-related effector proteins from bacteria to host cells [14]. Translocator proteins are involved in the trafficking of another set of proteins directly into the host cytoplasm through their needle like injectisome. After translocation, these "effector proteins" act as the virulence mediator affecting the changes in the host cells [15].
In this study, complete CRISPR/Cas9-based knockout ∆ssaU_SG18 strain was employed for a poultry experimental infection model to investigate the effect of the SPI-2 TTSS on the virulence of S. Gallinarum infections. Here, we demonstrate that a functional SPI-2 TTSS is necessary for the development of fowl typhoid and that ∆ssaU_SG18 is incapable of colonization and death in chickens.
The CRISPR system which was discovered as a bacterial immune system usually consists of the (CRISPR-associated) Cas protein (typically Cas9) (type IIA), an RNA-dependent endonuclease which cleaves the target DNA determined by the CRISPR RNA (crRNA) dictating the region of double stranded break (DSB) through its spacer sequence (20 nucleotide) and a trans-activating (tracrRNA) which forms a complex structure with crRNA to guide the Cas nuclease [16]. Nowadays this crRNA and tracrRNA can be fused together to transcribe a functional single chimeric guide RNA (sgRNA) [17], which form a complex with Cas9 protein directing it to specified DNA locus by base pairing with protospacer adjacent motif (PAM). PAM motif consists of a few base pairs with their specific sequence and position which vary with defined CRISPR/Cas9 system [18]. In the type II system of the S.pyogenes, PAM refers to a -NGG-consensus sequence comprising of two (G:C) base pairs which occur one base pair downstream of the spacer binding site, within the target DNA [16]. Hence after the recognition, this complex mediates a double stranded break (DSB), three base pairs upstream of the PAM sequence. Since, DSB is lethal, cells during their cycle of repair can be exploited to acquire specific modification via homologous recombination or integrated homology-directed repair (HDR) via a PCR product, plasmid or oligonucleotide [19].
Phage-derived lambda recombination system has emerged as a useful and alternate method for the chromosomal genome engineering [20,21]. The method relies upon the phage recombination proteins which utilize short homology region at 5 end of linearized DNA fragment to commence the homology directed recombination. The use of a phagederived recombination system has already been reported in many pathogenic organisms such as E.coli, Salmonella and Yersinia species [22][23][24]. More recently, the lambda red recombination based genome deletion was also reported in Salomella enterica serovar Pullorum in which the cigR deletion mutant was further tested for its virulence and biofilm formation [25]. The lambda red genes (i.e., gam, bet, and exo) from bacteriophage lambda are vital to support homologous recombination of linearized dsDNA [26]. Gam retards host endogenous nuclease activities of RecBCD and SbcCD; shielding the dsDNA for recombination apparently avoids the degradation of linear dsDNA template in the cell [27,28]. The Exo acts as an exonuclease which truncates linearized dsDNA in 5 →3 direction from both ends, forming 3 single-stranded tails for dsDNA [29]. Beta swiftly binds the ssDNA template ends to complementary strand to target DNA [30].
In this study, we have employed a simplified genome engineering strategy using CRISPR/Cas9 parallel with the conventional lambda red recombineering. This pairing increases the genome editing efficiency of the overall system with shorter time period required than previously reported methods [31]. An animal infection model was used to study the effect of ssaU mutant on the pathogenicity of this bacteria. This study demonstrates the versatility of CRISPR/Cas9 as a robust bioengineering tool for attenuated live vaccine development by targeting the different virulence-associated genes of pathogenic and resistant bacteria.

Strains, Culture Conditions, Plasmids, and Oligos
All the bacterial cultures along with the plasmids used in this study are mentioned in the Table 1. E. coli Top 10 cells used for transformation were cultured at 37 • C in Tryptic Soy Broth (TSB), supplemented with 100µg/mL of ampicillin or 50 µg/mL of kanamycin as required. S. Gallinarum (wild type) strain and transformants were grown aerobically at 37 • C in TSB supplemented with 50 µg/mL of kanamycin (kan), 100 µg/mL of ampicillin (amp) or chloramphenicol (cam) 25 µg/mL as per requirement. All the DNA oligonucleotide sequences used in this study are listed in Table 2. The linear DNA fragments designed for DSB repairing were amplified through Q5 ® High-Fidelity DNA Polymerase (NEB) after fusion using NEBuilder ® HiFi DNA Assembly as described in the Supplementary Material.

DNA Editing Template Construction, gRNA Cloning and Induction Method
Homology arms~1800 bp as DNA editing template were cloned in the pET22b (+) vector. Homology arms flanking up and downstream regions of the selected ssaU gene was amplified through overlap primers HA1_ssaU_Fp and HA1_ssaU_Rp for upstream while HA2_ssaU_Fp and HA2_ssaU_Rp for downstream region followed by fusion through NEB Hifi DNA ® assembly kit (see Supplementary Material). This assembly mixture was later amplified by using primer HA1_ssaU _Fp and HA2_ssaU_Rp and cloned in pET22b (+) vector named as RP-18. For dsDNA repair template, PCR product of fused homology arm fragments was used after PCR purification. In order to introduce targeted DSB, RNA spacer (5 -CGTTCCACTTCAAAAAATAA-3 ) and (5 -TCACGTAATTTCTTTTCTGT-3 ) were cloned in pCas9 and named as ssaU/G3, ssaU/G4, respectively. A Golden Gate assembly protocol was used for cloning of space sequence in plasmid (see Supplementary Material) [32,33]. As off target effects of gRNA has been reported, which could lead to secondary DSB or mutations, we used online available software Cas-OFFinder [34] (Bae et al., 2014) to rule out any secondary target site possibilities. Successful cloning of spacer sequence in pCas9 was confirmed by using primers Cas9_R as reverse and gRNA3F_ssaU or gRNA4F_ssaU as forward primers for ssaU/G3 and ssaU/G4, respectively ( Figure 1). Spacer cloning was confirmed by Sanger sequencing (Supplementary Material Figure S1). Mutants were identified using screening primers designed outside of the frame of homology arm sequence named ssaU _screening_Fp and ssaU _screening_Rp. Mutants were further confirmed by sanger sequencing.
In all of the experiments exhibiting recombineering, controls were kept in place to assess the electroporation of plasmids. At least three replicates were employed to assess the recombination of the plasmid/dsDNA template for each recombineering experiment performed. The formula for calculating transformation and electroporation efficiency is provided in the Supplementary Material. Statistical Analysis including arithmetic means Mutants were identified using screening primers designed outside of the frame of homology arm sequence named ssaU _screening_Fp and ssaU _screening_Rp. Mutants were further confirmed by sanger sequencing.
In all of the experiments exhibiting recombineering, controls were kept in place to assess the electroporation of plasmids. At least three replicates were employed to assess the recombination of the plasmid/dsDNA template for each recombineering experiment performed. The formula for calculating transformation and electroporation efficiency is provided in the Supplementary Material. Statistical Analysis including arithmetic means with SD and p-value using two-way ANOVA was performed using the software tool Graphpad prism version 8.0.1 GraphPad Software, La Jolla, CA, USA, www.graphpad.com (accessed on 20 July 2022).

Mutant Strain Preparation
The red recombineering plasmid (pRed) was electroporated into the S. Gallinarum cells selected on 50 µg/mL kanamycin and confirmed through colony PCR using Pcasred-Fp and Pcas-red-Rp as forward and reverse primers, respectively. Overnight cultures of the recombineering plasmid transformed S. Gallinarum cells (500 µL) were shifted to 30 mL TSB at 30 • C for~1.5 h with shaking at 200 rpm. When OD 600 nm of cells reached 0.2, the cells were induced by 10 mM arabinose for expression of lambda recombineering components (Exo, Gam, Beta) and further incubated at 30 • C for~1 h with shaking. These cells were then used for electrocompetent cells preparation.

Electroporation
A single colony of the wild type S. Gallinarum was inoculated in 3 mL TSB overnight. Overnight culture from this single colony was diluted (500 µL) in 50 mL of the TSB and incubated at 37 • C with shaking at 200 rpm. When OD 600 of the cells reached 0.2-0.3, the cells were subjected to chilled ice for~10 min and later centrifuged at 5000 rpm for 5 min at 4 • C. The supernatant was discarded and cells were washed with 20 mL sterile distilled water once and twice with sterile 10% glycerol solution at 4 • C. Finally, the cells were resuspended in 1 mL 10% glycerol solution and 100 uL aliquots were stored at −80 • C.
According to electroporation requirements, aliquots were thawed on ice for 10 min. Then the cells were mixed with 8 µg of the required plasmid/linearized DNA and transferred to pre-chilled 2 mm electroporation cuvette (Bio-Rad). Cells were then pulsed at 25 KV/cm, 1100 Ω, and 25 µF, with time constant of 5.0 milli-second. Immediately after the pulse, cells were mixed with the TSB (1 mL) and incubated at 37 • C for~2 h, which were then plated on TSA plates containing corresponding antibiotics. Plates were incubated at 37 • C for 24-36 h or until the colonies were evident. Mutants were screened using cPCR.

Poultry Experimental Model to Evaluate Virulence
To infect broiler birds, wild type S. Gallinarum (WT-SG18) and modified (∆ssaU_SG18) were used. A total of 200 disease-free male broiler chickens of the Cobb 500 strain were used in this study which were procured from a commercial hatchery. The birds were kept in sterile pens in an environmentally-controlled room with a 20-h light cycle. The birds were distributed randomly into four groups: SG_WT (S. Gallinarum positive control), SG_Negative (not infected with any pathogens), complement strain (cSG18) and ∆ssaU_SG18 mutant group, with five equal sized replicates containing 10 birds each on the first day. On day 7, cloacal swabs were used to randomly sample birds for the screening of SG infection. Throughout the duration of the experiment, the birds had unfettered access to antibiotic-free food and water. At 16 days of age, birds were administered 1 mL of normal saline containing approximately 1 × 10 8 colony-forming units (CFU) of each strain via oral gavage. Birds were fed orally with SG in order to mimic the natural route of infection. After administration, birds were routinely observed for gross signs of disease. Throughout the trial, birds were weighed each week to record any changes in weight gain. Gross examination and post-mortem investigations of both dead and critically ill birds were conducted 21 days post-infection.

Lethality of Cas9 Induced Double Stranded Break and Efficiency of Repair Plasmids
We employed multiple techniques for optimizing CRISPR/Cas9-mediated genome editing in the SG, by targeting the selected ssaU gene encoding type three secretion system apparatus (TTSS), which is located on Salmonella pathogenicity island 2 (SPI-2) comprising 1059 nucleotides. Initially, two plasmid approach was employed in which the modified pCas9 plasmid [35] containing the constitutively expressing Cas9 nuclease for DSB and repair template plasmid RP-18 was used.
Co-electroporation of the modified pCas9 ssaU/G3 and RP-18 (DNA editing plasmid) into SG cells resulted in no clones. However, colonies consistently appeared on the control plate when electroporated with simple pCas9 plasmid (with no guide RNA) and RP-18, with transformation efficiency of (8.2 × 10 2 ± 25 cfu/µg n = 3), which rule out the toxic effect of empty Cas9 nuclease and suggest the lethality of Cas9 nuclease due to cloned gRNA causing genomic DSB [32]. Clones were not evident even after utilizing a higher amount (10 µg) of repair template plasmid or decreasing the ssaU/G3 (< 50 ng) concentration.
This suggested that the DSB induced by ssaU/G3 was not resolved by the homologydirected repair, meanwhile native non-homologous end joining (NHEJ) was also not efficient in the SG [36]. We further cloned a second spacer RNA in pCas9 (ssaU/G4) targeting another region of the ssaU gene (Figure 1), however, no colonies were observed after electroporation with the plasmid based homology templates RP-18. A two-step electroporation was also performed, where RP-18 was electroporated first into SG to allow the cross-over event to occur before the electroporation of ssaU/G3 or ssaU/G4. However, this strategy still yielded no clones.

Coupling CRISPR/Cas9 with Lambda Red Recombineering Results in Successful Gene Knock Out
To overcome the lower recombination capacity of the bacterial cells after DSB, we used the lambda red recombineering plasmid pRed available in our lab amplified from pCas in combination with pCas9 [37]. After the induction with 10 Mm arabinose and followed by the co-electroporation with ssaU/G3 (300 ng) and linearized dsDNA repair template (8 µg) clones were apparent on the selection plate. The colonies that appeared were screened through colony PCR. Surprisingly a maximum of (70 ± 2.5% n = 3) clones showed successful deletion of the said gene. The PCR screens of the ssaU gene deletion is shown in (Figure 2), unraveling the high potential of this unified system for efficient gene deletion in S. Gallinarum.
Deletion of the ssaU gene was further confirmed through Sanger sequencing (Supplementary Material Figure S2), whereas no mutation was observed in the flanking upstream and downstream regions of the deleted gene confirming the precise genome modification.

Clearance of ssaU Mutant Strain from Experimental Birds
In order to determine the systemic infection, bacteria were isolated from visceral organs of the birds, primarily the liver and ileum, after 4, 7 and 14 days post-infection. The WT-SG18 strain recovered from the liver and intestine of infected birds (positive control group) showed 1.3 × 10 3 , 5.0 × 10 5 and 1.6 × 10 8 CFU/g after 4, 7 and 14 days, respectively, postinfection indicating systemic bacteremia. Our results are in agreement with the previously reported study showing the highest number of viable counts that were isolated from the liver after 14 days post-infection with S. Gallinarum [38]. In contrary, ∆ssaU_SG18 strain could not be recovered from any of the tissues, even following enrichment with selenite broth, although it was recovered from the caecum 2 days post-infection. The post-mortem examination of WT-SG18 group exhibited the typical signs of fowl typhoid pathology (Figure 3), including hepatosplenomegaly with necrotic foci on liver and spleen, bronze discoloration of liver and hemorrhages in the anterior small intestine. In contrast, the ∆ssaU_SG18 strain showed no signs and symptoms of disease at postmortem examination throughout the period of experiment (Figure 3).

Knock Out
To overcome the lower recombination capacity of the bacterial cells after DSB, we used the lambda red recombineering plasmid pRed available in our lab amplified from pCas in combination with pCas9 [37]. After the induction with 10 Mm arabinose and followed by the co-electroporation with ssaU/G3 (300 ng) and linearized dsDNA repair template (8 μg) clones were apparent on the selection plate. The colonies that appeared were screened through colony PCR. Surprisingly a maximum of (70 ± 2.5% n = 3) clones showed successful deletion of the said gene. The PCR screens of the ssaU gene deletion is shown in (Figure 2), unraveling the high potential of this unified system for efficient gene deletion in S. Gallinarum. Step (2) co-electroporation of (ssaU/G3) along with RP-18 DNA editing plasmid in SG does saU_SG18 strain could not be recovered from any of the tissues, even following enrichment with selenite broth, although it was recovered from the caecum 2 days post-infection. The post-mortem examination of WT-SG18 group exhibited the typical signs of fowl typhoid pathology (Figure 3), including hepatosplenomegaly with necrotic foci on liver and spleen, bronze discoloration of liver and hemorrhages in the anterior small intestine. In contrast, the ΔssaU_SG18 strain showed no signs and symptoms of disease at postmortem examination throughout the period of experiment (Figure 3). Our results suggest that the avirulent strain ∆ssaU_SG18 was unable to invade from the intestine to the liver, which is consistent with a previously reported work where the avirulent ssaU mutant of S. Gallinarum was not recovered from tissues of birds, hence suggesting their rapid clearance after infection [40]. The particular loss of SPI-2 function in serovar Typhimurium leading to attenuation of typhoidal disease in mice models has been previously reported [41]. The complement strain (cSG18) which was constructed using pTZ19R to achieve continuous expression of ssaU showed virulence identical to that of WT-SG18, further confirming the role of this gene in the pathogenicity of bacteria. Weight gain and progression of mortality for both WT-SG18, negative control and ∆ssaU_SG18 during the period of experiment is shown in (Figure 5). The final weight gain of ∆ssaU_SG18 and negative control revealed no significant difference (p-value = 0.8166), whereas the WT-SG18 group exhibited higher significance (p-value < 0.0001) due to disease progression and systemic infection leading to abrupt loss in weight gain. The overall mortality data also showed higher significance (p-value < 0.0001), where no mortality was observed in the ∆ssaU_SG18 group, while the WT-SG18 group demonstrated an overall mortality of 32 ± 4.4% starting at day 6 ( Figure 5B).
Histopathological analysis of the liver, spleen and intestine revealed lesions showing marked necrosis, hemorrhages, congestion and infiltration of heterophils (avian polymorphonuclear cells) characteristic of fowl typhoid (Figure 4) [39]. Our results suggest that the avirulent strain ΔssaU_SG18 was unable to invade from the intestine to the liver, which is consistent with a previously reported work where the avirulent ssaU mutant of S. Gallinarum was not recovered from tissues of birds, hence suggesting their rapid clearance after infection [40]. The particular loss of SPI-2 function in serovar Typhimurium leading to attenuation of typhoidal disease in mice models has been previously reported [41]. The complement strain (cSG18) which was constructed using pTZ19R to achieve continuous expression of ssaU showed virulence identical to that of WT-SG18, further confirming the role of this gene in the pathogenicity of bacteria. Weight gain and progression of mortality for both WT-SG18, negative control and Δs-saU_SG18 during the period of experiment is shown in (Figure 5). The final weight gain of ΔssaU_SG18 and negative control revealed no significant difference (p-value = 0.8166), whereas the WT-SG18 group exhibited higher significance (p-value < 0.0001) due to disease progression and systemic infection leading to abrupt loss in weight gain. The overall mortality data also showed higher significance (p-value < 0.0001), where no mortality was

Discussion
For the past 20 years, genetic engineering using recombineering has been employed as a robust tool to modify the bacterial genome [42]. More recently, the CRISPR/Cas9 system has highlighted its potential also as a powerful tool for the genetic manipulations and hence revolutionizing genome engineering. Therefore, we have demonstrated the use of

Discussion
For the past 20 years, genetic engineering using recombineering has been employed as a robust tool to modify the bacterial genome [42]. More recently, the CRISPR/Cas9 system has highlighted its potential also as a powerful tool for the genetic manipulations and hence revolutionizing genome engineering. Therefore, we have demonstrated the use of such a coupled system to modify the genome of SG which provides a streamlined approach for knock-out mutant generation. The first approach to genetically manipulating the SG genome using two plasmids (ssaU/G3 and RP18) did not produce any mutant clones. We hypothesized that the plasmid-based DNA editing template was less efficient. The lower efficiency of plasmid-based repair templates has already been reported in the previous studies [43].
Considering this, we moved on to the lambda red recombineering proteins which have been proven to be influential in enhancing the recombination capacity of bacteria [44,45]. By using the SG cells expressing lambda protein along with ssaU/G3 and dsDNA template. The recombination efficiency in case of gene knockout was 70 ± 2.5% using the CRISPR/Cas9 and recombineering system together. We were unable to obtain any clone using only lambda recombination alone and not CRISPR/Cas9 system. The plasmid-based homologous recombination using CRISPR/Cas9 causes the vector integration event (VIE) problems (homologous recombination by double crossover), which further needs screening steps for the selection of the required positive mutant [17,32]. Additionally, the lambda red recombination alone depended upon the integration of the resistant marker, hence this coupled methodology provides a straightforward design for S. Gallinarum genome editing that too with higher efficiency. As this approach does not require construction of HDR plasmid, it provides a reliable methodology for precise knock-out production.
A complete deletion of the ssaU gene from S. Gallinarum resulted in in vivo attenuation; however, further deletion of other virulent genes from the ∆ssau_SG18 strain will make this strain a suitable candidate for vaccine design. TTSS is required for triggering apoptosis in the epithelial cells [46]. In both apoptosis (enteric phase) and pyroptosis (delayed macrophage death), TTSS is instrumental in the translocation of effectors into the host cell during the early phase of pathogenesis [47], whereas the effector proteins (SpvB, SseL, SlrP), which are crucial for host cellular dysfunction and deterioration, are expressed in the later stages of infections [48,49]. Several TTSS components have been found to be crucial for SpvB-induced actin-depolymerization and cellular cytotoxicity, lending support to the fact that SpvB is translocated into the host cell via TTSS [50,51]. The role of SpvB as potential virulent effector protein has previously been identified in our lab where SpvB mutant of SG was also unable to produce disease symptoms in the poultry infection model [52]. Hence, it can be speculated that the TTSS act as a vital machinery to deliver the effector proteins inside the host cell which give rise to the induction of virulence. Here we have highlighted the significance of the SPI-2 TTSS in a naturally occurring route of systemic, typhoid-like Salmonella infection that has economical and veterinary significance in developing countries. Following this optimized CRISPR/Cas9-based manipulation, studies are underway in our lab to produce an indigenous vaccine strain by deletion of a larger pathogenicity island to control this disease which has been a major concern for Pakistan poultry industry.

Conclusions
This study shows the importance of the SPI-2 TTSS in the infection of typhoid-like salmonellosis that has significant economic and veterinary importance in under developed countries. The SPI-2 TTSS allows the serovar Gallinarum to cause the disease by facilitating the bacterial survival within the macrophages and reticuloendothelial system. This coupled CRISPR/Cas9 and lambda recombineering system can be formulated for the live vaccine development against indigenous S. Gallinarum by large-scale deletion of Salmonella pathogenicity islands or relative virulence genes [32].

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10123028/s1, Figure S1: Confirmation of gRNA cloning by Sanger sequencing., Figure S2: Confirmation of precise gene knock-out by Sanger sequencing, Calculation of transformation and recombination efficiency, Detailed protocol for Genome engineering of Salmonella Gallinarum.