Development and Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus

Lactobacillus, a genus of lactic acid bacteria, plays a crucial function in food production preservation, and probiotics. It is particularly important to develop new Lactobacillus strains with superior performance by gene editing. Currently, the identification of its functional genes and the mining of excellent functional genes mainly rely on the traditional gene homologous recombination technology. CRISPR/Cas9-based genome editing is a rapidly developing technology in recent years. It has been widely applied in mammalian cells, plants, yeast, and other eukaryotes, but less in prokaryotes, especially Lactobacillus. Compared with the traditional strain improvement methods, CRISPR/Cas9-based genome editing can greatly improve the accuracy of Lactobacillus target sites and achieve traceless genome modification. The strains obtained by this technology may even be more efficient than the traditional random mutation methods. This review examines the application and current issues of CRISPR/Cas9-based genome editing in Lactobacillus, as well as the development trend of CRISPR/Cas9-based genome editing in Lactobacillus. In addition, the fundamental mechanisms of CRISPR/Cas9-based genome editing are also presented and summarized.


Introduction
Lactobacillus is a genus of rod-shaped, non-spore-forming, Gram-positive bacteria that produce large amounts of lactic acid from fermentable carbohydrates [1]. Lactobacillus belongs to Firmicutes, Bacilli, Lactobacillales, Lactobacillaceae, which is an important genus of the Lactobacillaceae [2]. Lactobacilli are found in plants and plant products, silage, dairy products, fermented foods, as well as in the oral cavity, vagina, and gut of humans and animals [3]. Lactobacilli are not only one of the symbiotic bacteria in the human body, but they are also essential to human health and the food industry.
Lactobacilli are generally recognized as safe (GRAS) microorganisms and have a wide range of industrial applications [4,5]. The application of Lactobacillus in food fermentation has a long history and it can be used to produce fermented dairy products, meat, and vegetable food, such as yogurt, cheese, sausage, and pickles [6][7][8][9]. Lactobacilli utilize fermentable saccharides to produce mild sour lactic acid, and they can also produce other organic acids, such as propionic acid, formic acid, and acetic acid, which constitute the main flavor components of food [10]. While giving the product a sour taste, they can also interact with alcohols, aldehydes, ketones, and other substances produced in the process of lactic acid fermentation to form a variety of new flavoring substances, which constitute the aromatic components in food [11]. Lactobacillus can also be used as probiotics, with a variety of prebiotic functions ( Table 1). The global probiotic market is anticipated to reach~92 billion USD in sales by 2026, and the probiotic supplement 2 of 15 industry is anticipated to grow at a compound annual growth rate (CAGR) of over 8% between 2021 and 2026 [12]. Therefore, Lactobacillus, as a potential candidate for probiotics, has substantial economic significance.
Gene editing is a revolutionary genetic engineering technique that precisely modifies particular target genes in the genomes of organisms [13]. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-related protein (Cas) system [14] has undergone constant development over the past few years, and is now in its third generation. In comparison to the first-generation Zincfinger Nucleases (ZFNs) and the secondgeneration Transcription Activator-like Effector Nucleases (TALENs), the CRISPR/Cas9 technology is the most effective and feasible and the least expensive [13,15,16]. The CRISPR/Cas system is an acquired immune system that prokaryotes utilize to resist the invasion of exogenous genetic elements present in bacteriophages or plasmids, and it is a defense mechanism present in most bacteria and all archaea to eliminate alien plastid or phage DNA (Figure 1) [17]. The core of this system consists of two parts: CRISPR sequence and Cas gene [18]. CRISPR is a series of tandem repetitive DNA fragments accidentally found in the genome of E. coli [19]. Subsequently, it was found that such repetitive sequences are widely found in the genome of prokaryotes and archaea, and they were named as Clustered Regularly Interspaced Short Palindrome (CRISPR) sequences, and Cas is a related protein with nuclease activity [20,21]. The RNA corresponding to the spacer in CRISPR sequence can be used to identify and cut specific complementary DNA strands [22]. The CRISPR/Cas systems are divided into two classes and six types based on the structure and function of the Cas protein. Class I includes types I, III, and IV, and class II includes types II, V, and VI. The CRISPR/Cas9 system belongs to the type II system, and has developed rapidly as the widely used tool in the precise genome editing [23][24][25]. Cholesterol-lowering activity [32] Currently, CRISPR/Cas9 technology is utilized infrequently on Lactobacillus. Utilizing CRISPR/Cas9 for effective gene editing of Lactobacillus to strengthen strains with prebiotic properties or to introduce additional prebiotic functionalities as needed would expand the application of Lactobacillus in food development, disease treatment, and nutritional wellness. This review will summarize the development and applications of CRISPR/Cas9-based genome editing in Lactobacillus over the past few years, and advances and applications of CRISPR/Cas9 technology and its prospects for this genus will be described. Figure 1. CRISPR/Cas9 system in bacterial adaptive immunity. The functional driving of CRISPR/Cas9 system is divided into three stages. Acquisition: After phage infection, the Cas1-Cas2 protein complex will scan the invading DNA and identify the PAM region, and integrate the new spacer into the CRISPR sequence through the collection mechanism (cas1, cas2, csn2). Transcription: CRISPR sequence is transcribed to produce pre-crRNA under the control of the leading region, and tracrRNA is also transcribed. Pre-crRNA is further processed to form mature crRNA, which forms double-stranded RNA with tracrRNA through base complementary pairing. (3) Interference: The crRNA-tracrRNA duplex binds to Cas9 and guides Cas9 to cleave foreign DNA containing a 20-nt crRNA complementary sequence preceding PAM.

Components of CRISPR/Cas9 Genome Editing System
Cas9, crRNA, and tracrRNA are the three components that comprise the natural CRISPR/Cas9 system [33]. Through local base pairing, crRNA and tracrRNA form guide RNA (gRNA), and gRNA binds to Cas9 protein to guide Cas9 protein to specifically recognize and cleave target DNA sequences (Figure 2A) [34]. Researchers fused crRNA and tracrRNA into one RNA, and called it single guide RNA (sgRNA) in order to facilitate experimental design and improve the stability of gRNA ( Figure 2B) [35]. The modified CRISPR/Cas9 system has become the preferred tool for researchers for gene editing. For Cas9 protein to successfully recognize the target sequence, two conditions must be met: (1) base pairing between 20 NT at the 5'-end of sgRNA and target DNA; (2) presence of an appropriate PAM sequence at the 3'-end of target DNA. In the CRISPR/Cas9 system, the Cas9 protein contains HNH and RuvC domains that cleaves target DNA to generate DNA double-strand breaks (DSBs) [36,37]. The sgRNA can be engineered to recognize upstream sequences to PAM (NGG); therefore, the CRISPR/Cas9 system has increasingly become an accessible tool. . CRISPR/Cas9 system in bacterial adaptive immunity. The functional driving of CRISPR/Cas9 system is divided into three stages. Acquisition: After phage infection, the Cas1-Cas2 protein complex will scan the invading DNA and identify the PAM region, and integrate the new spacer into the CRISPR sequence through the collection mechanism (cas1, cas2, csn2). Transcription: CRISPR sequence is transcribed to produce pre-crRNA under the control of the leading region, and tracrRNA is also transcribed. Pre-crRNA is further processed to form mature crRNA, which forms double-stranded RNA with tracrRNA through base complementary pairing. (3) Interference: The crRNA-tracrRNA duplex binds to Cas9 and guides Cas9 to cleave foreign DNA containing a 20-nt crRNA complementary sequence preceding PAM.

Mechanism of CRISPR/Cas9 Genome Editing System
Gene editing technology based on CRISPR/Cas9 is mainly divided into two pr (Figure 3), i.e., DNA cleavage and DNA repair [38].
DNA cleavage: Cas9 is recruited by guide RNA and binds to guide RNA to complex, Cas9 nuclease activity is activated, and the formed complex can start to l the complementary target DNA sites for recognition and cleavage, and the Cas9 specifically cleaves the target DNA sequence to generate double-strand breaks [39,40]. Target recognition and cleavage requires not only complementary pairing original 20-nt spacer sequence in the target DNA and the spacer on the guide RN also the existence of a conserved PAM motif in the target attachment [41]. A single tion in PAM in vitro can lead to loss of Cas9 cleavage activity and enable phages to host immune responses [42]. The commonly used SpCas9 (Cas9 nuclease from Stre cus pyogenes) PAM sequence is 5'-NGG-3', where N can be any of the four DNA ba DNA repair: This mechanism in the cell repairs the DSBs and modifies the D quence during the repair process [43]. DNA repair mechanisms are divided into tw egories: homology-directed repair (HDR) and non-homologous end joining (NHE HDR requires the help of repair templates, which can achieve accurate and contr editing; NHEJ repair does not rely on repair templates and directly splices two DN [45][46][47]. However, base insertion or deletion (indels) may occur during the splicin cess, and accurate editing cannot be achieved. In Lactobacillus, the introduced DS usually repaired by HDR, not NHEJ [48].

Mechanism of CRISPR/Cas9 Genome Editing System
Gene editing technology based on CRISPR/Cas9 is mainly divided into two processes (Figure 3), i.e., DNA cleavage and DNA repair [38]. Figure 3. Mechanism of CRISPR/Cas9 genome editing system. The guide RNA directs a nuclease to cleavage target DNA. The DSB (double-strand break) generated by Cas9 nu mains is repaired by host-mediated DNA repair mechanisms, i.e., homology-directed re and non-homologous end joining (NHEJ).

Modifications of Cas9 for Genome Editing
Cas9 proteins with nuclease activity are found in the majority of bacteria. Th produced from Streptococcus pyogenes is the most commonly utilized Cas9 for ge DNA cleavage: Cas9 is recruited by guide RNA and binds to guide RNA to form a complex, Cas9 nuclease activity is activated, and the formed complex can start to look for the complementary target DNA sites for recognition and cleavage, and the Cas9 protein specifically cleaves the target DNA sequence to generate double-strand breaks (DSBs) [39,40]. Target recognition and cleavage requires not only complementary pairing of the original 20-nt spacer sequence in the target DNA and the spacer on the guide RNA, but also the existence of a conserved PAM motif in the target attachment [41]. A single mutation in PAM in vitro can lead to loss of Cas9 cleavage activity and enable phages to evade host immune responses [42]. The commonly used SpCas9 (Cas9 nuclease from Streptococcus pyogenes) PAM sequence is 5'-NGG-3', where N can be any of the four DNA bases.
DNA repair: This mechanism in the cell repairs the DSBs and modifies the DNA sequence during the repair process [43]. DNA repair mechanisms are divided into two categories: homology-directed repair (HDR) and non-homologous end joining (NHEJ) [44]. HDR requires the help of repair templates, which can achieve accurate and controllable editing; NHEJ repair does not rely on repair templates and directly splices two DNA ends [45][46][47]. However, base insertion or deletion (indels) may occur during the splicing process, and accurate editing cannot be achieved. In Lactobacillus, the introduced DSBs are usually repaired by HDR, not NHEJ [48].

Modifications of Cas9 for Genome Editing
Cas9 proteins with nuclease activity are found in the majority of bacteria. The Sp-Cas9 produced from Streptococcus pyogenes is the most commonly utilized Cas9 for gene editing [49]. Cas9 protein contains two characteristic nuclease domains ( Figure 4A): HNH domain and RuvC domain, inducing DSBs in the target DNA, where the RuvC domain cleaves the non-complementary strand of DNA and the HNH domain cleaves the DNA strand that is complementary to crRNA [50]. The intensity of nuclease activity depends largely on the binding efficiency of Cas9 [51]. Subsequently, some Cas9 mutants emerged to improve gene editing efficiency [52].
Ding et al. demonstrated that SpCas9 could be modified by fusing with chromatin regulatory peptides (CMPs) from high mobility group proteins HMGN1 and HMGB1, histone H1, and the chromatin remodeling complex, and that its activity could be multiplied by several orders of magnitude, particularly on refractory targets [53]. Additionally, the CMPs fusion strategy (CRISPR chrome) is also effective in enhancing the activity of the newly characterized Cas9 homolog of L. rhamnosus. At the same time, this CRISPR chrome strategy can be used to improve the established Cas9 nuclease and encourage the discovery of new Cas9 homologues for genome modification.
Cas9 nickase (Cas9n) consists of inactivating mutations in the endonuclease domain [54]. For instance, the Cas9 D10A mutation eliminates the endonuclease activity of the RuvC domain while retaining one HNH domain function ( Figure 4B); likewise, the Cas9 H840A mutation can eliminate the function of the HNH domain while retaining the RuvC function ( Figure 4C), which is capable of forming single-strand break (SSB) at the target position [54][55][56]. Goh et al. applied the gene editing method in L. acidophilus using the established Cas9 D10A nickase variant (Cas9n) [57]. The obtained pLbCas9 N system has been further demonstrated in L. gasseri and L. paracasei. The system (pLbCas9n) has high gene editing efficiency and versatility. It can function flexibly at different chromosomal loci, generate deletions of varying sizes, and construct repair templates for many concurrent mutations. Deletion mutants can be recovered within one week after transformation. Additionally, in some cases, edited cells are present with unedited cells, and subsequent purification can be applied to mixed genotype cultures to recover pure mutant populations.
lished Cas9 D10A nickase variant (Cas9n) [57]. The obtained pLbCas9 N system has been further demonstrated in L. gasseri and L. paracasei. The system (pLbCas9n) has high gene editing efficiency and versatility. It can function flexibly at different chromosomal loci, generate deletions of varying sizes, and construct repair templates for many concurrent mutations. Deletion mutants can be recovered within one week after transformation. Additionally, in some cases, edited cells are present with unedited cells, and subsequent purification can be applied to mixed genotype cultures to recover pure mutant populations.

Guide RNA Expression
The specificity of the CRISPR/Cas9 system for DNA recognition is not determined by proteins, but by 20nt guide RNA [39,58]. By artificially designing crRNA and tracrRNA, sgRNA (single guide RNA), with a guiding effect, can be transformed to guide Cas9 to cleave DNA at the target region, and the rational design of sgRNA can effectively reduce the off-target effects [59][60][61]. At the constant expression level of Cas9 nuclease, the amount of sgRNA transcription was positively correlated with the cleavage efficiency of Cas9 sgRNA complex on DNA. Therefore, selecting strong promoters to enhance the amount of sgRNA transcription can improve gene editing efficiency [62]. Promoter P11 is a synthetic sequence of rRNA promoter from L. plantarum WCFS1, which is a strong constitutive promoter. Studies have shown that replacing the strong promoter P11 with the promoter PLP _0537 can enhance the amount of sgRNA transcription, hence improving the editing efficiency of L. plantarum WCFS1 [63].

Guide RNA Expression
The specificity of the CRISPR/Cas9 system for DNA recognition is not determined by proteins, but by 20nt guide RNA [39,58]. By artificially designing crRNA and tracrRNA, sgRNA (single guide RNA), with a guiding effect, can be transformed to guide Cas9 to cleave DNA at the target region, and the rational design of sgRNA can effectively reduce the off-target effects [59][60][61]. At the constant expression level of Cas9 nuclease, the amount of sgRNA transcription was positively correlated with the cleavage efficiency of Cas9 sgRNA complex on DNA. Therefore, selecting strong promoters to enhance the amount of sgRNA transcription can improve gene editing efficiency [62]. Promoter P11 is a synthetic sequence of rRNA promoter from L. plantarum WCFS1, which is a strong constitutive promoter. Studies have shown that replacing the strong promoter P11 with the promoter PLP _0537 can enhance the amount of sgRNA transcription, hence improving the editing efficiency of L. plantarum WCFS1 [63].
In most cases, phase recombinase is required for recombineering. However, investigations have demonstrated that phase recombinase is not required [48]. Leenay et al. used CRISPR/Cas9-assisted ssDNA recombineering, exogenous recombinase-free genome editing to simplify CRISPR/Cas9-assisted ssDNA recombineering [67]. This approach enabled the editing of three genes in L. plantarum WJL, including the addition of a presequence stop codon in the ribB gene associated with vitamin B2 production and the introduction of multiple point mutations in the ackA gene encoding acetate kinase. The lacM gene involved in β-galactosidase production was removed to examine whether this method produces additional mutations in other genes of L. plantarum WJL and to determine whether this technique may produce entire gene deletions. This exogenous recombinase-free technique simplifies L. plantarum WJL genome editing.
Vento et al. utilized two E. coli-Lactobacilli shuttle vectors: one containing SpCas9, tracr-RNA, and single spacer CRISPR array; the other containing a dsDNA recombinant engineering template [69]. L. plantarum was transformed with an E. coli-Lactobacillus shuttle vector carrying a recombineering template (RT) and homologous arm (HA), and L. plantarum was edited using this technique. It is a straightforward and rapid approach of gene editing. Before the correct mutant can be screened, genome editing requires two consecutive transformations of Lactobacillus, which can be accomplished in 10 days.

RecE/T-Assisted CRISPR/Cas9 System
The RecE/T recombination system was found in E. coli and encoded by RecE and RecT genes [70]. RecE is a 5 -3 dsDNA exonuclease that cleave exogenous dsDNA to generate a 3 -ended single stranded DNA overhang, and RecT is a single-strand annealing protein (SSAP) that binds to the ssDNA overhangs and promotes strand exchange and strand invasion [71,72]. Huang et al. coupled phage derived RecE/T with CRISPR/Cas9, and established a universal toolbox containing recombinant auxiliary plasmids and broadspectrum host CRISPR/Cas9 editing plasmids [63]. This toolbox can perform effective genome editing in L. plantarum WCFS1 and L. brevis ATCC367. The RecE/T-assisted CRISPR/Cas9 toolbox achieves single gene deletion with 50-100% efficiency in seven days.

Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus
As whole genome sequencing technology has advanced, it has been discovered that certain genes in the Lactobacillus genome have important functions. These functions include the metabolism of sugars, the production of bacteriocin and extracellular polysaccharides, protein hydrolysis, and phage resistance, etc. [72,73]. Besides, Lactobacillus has good resistance to an adverse environments; thus, Lactobacillus can be used as a cell factory for biological purification and production of bioactive compounds [74]. In addition, several Lactobacilli have demonstrated probiotic properties. CRISPR/Cas9-based genome editing technology is used to develop improved strains with the intent of enhancing the properties of Lactobacilli, enhancing the quality and quantity of products, or enhancing and expanding probiotic functions.
Recently, CRISPR/Cas9-based genome editing technology was applied to several Lactobacilli (Table 2). Lactobacillus can produce two kinds of lactic acid at the same time, L-lactic acid and D-lactic acid. D-lactate dehydrogenase gene (ldhD) and L-lactate dehydrogenase gene (ldhl) are the key genes that regulate the production of these two types of lactic acid [75]. Tian et al. knocked-out the ldhD gene and introduced the ldhL1 gene through CRISPR/Cas9 genome editing system to transform L. paracasei into a high optical purity L-lactic acid producing strain, which could efficiently produce L-lactic acid under 45 • C growth condition [76]. Wang et al. developed single, double, and triple bsh knock-out strains using CRISPR/Cas9 genome editing technology in order to determine the significance of different bsh genes in bile salt resistance of L. plantarum AR113 [77]. The results demonstrated that bsh1 and bsh3 are closely associated with bile salt resistance and will aid in the selection of strains with a high tolerance for bile salts.
To prevent Lactobacillus from dying following the formation of DSBs, Goh et al. developed a Cas9 nickase-based genome editing technique that reduces strain mortality from double-strand breaks (DSBs) in L. acidophilus, L. casei, and L. gasseri [57]. To prevent cell death caused by DSBs, Song et al. developed a rapid and precise gene editing method in L. casei, utilizing Cas9 nickase that only cleaves a single-strand of DNA, with knock-out and knock-in efficiencies ranging from 25% to 62% [78]. Using this method, the researchers eliminated four non-essential genes and inserted a green fluorescent protein reporter gene. The current gene editing technique for L. casei relies on plasmid-based homologous recombination, which requires at least 24 days to produce one gene knock-out. The CRISPR/Cas9 D10A system decreased the cycle time to nine days. Li et al. utilized the CRISPR/Cas9 D10A system to create auxotrophic Lactobacillus W56 (Lactobacillus ∆Alr W56) and auxotrophic L. paracasei HLJ-27 (Lactobacillus ∆Alr HLJ-27) [79]. By inserting the VP4 gene into the genome using a temperature-sensitive gene editing plasmid, the insertional mutant strains ∆VP4/Alr HLJ-27, ∆VP4/Alr W56, and VP4/W56 were subsequently created. The VP4 protein, which is encoded by the VP4 gene, is an effective antigen for the development of an anti-PoRV (Porcine rotavirus) vaccine. After oral immunization of mice with the recombinant strains, there were significantly higher levels of serum IgG (immunoglobulin G) and mucosal SIgA (secretory immunoglobulin A), indicating that the insertional mutant strains ∆VP4/Alr HLJ-27, ∆VP4/Alr W56, and VP4/W56 were able to induce the production of mucosal and humoral immune responses against PoRV (Porcine rotavirus).
Zhou et al. [68] discovered that encoding DNA adenine methylase (Dam) in L. plantarum WCFS1 improved the efficiency of CRISPR/Cas9-assisted ssDNA recombination by further optimizing the point mutation method. The introduction of Dam increased the efficiency of point mutation for the L. lantarum glmS1 gene to 71.4%. Stout et al. identified the function of the CRISPR/Cas9 system in L. gasseri JV-V03 and L. gasseri NCK1342 and initially noticed the existence of escapees for the off-target effect of Cas9 plasmid transformation [80]. The mechanism of CRISPR-targeted escape was subsequently investigated in further detail. Plasmid interference tests were used to explore the targeting and escape mechanisms of CRISPR/Cas9 system, and the results showed that spacer deletions are the primary escape mechanism in CRISPR/Cas9 system. This research will contribute to a better understanding of the primary causes behind the occasional targeting failures of the CRISPR/Cas9 system.

CRISPR/Cas9-Based Transcriptional Regulation in Lactobacillus
The CRISPR/Cas9 technology can regulate gene expression in addition to being utilized for genome editing. Dead Cas9 (dCas9), nuclease-null Cas9, inhibits the enzymatic activity of RuvC and HNH domains in Cas9 nuclease ( Figure 4D) [82]. However, it can still bind to the double strand, hindering the binding of RNA polymerase to the target gene and becoming an important tool for gene silencing [83]. The dCas9 system has been developed as a tool that enabled transcription regulation, epigenetic modifications, DNA looping, and genome imaging [84][85][86]. Based on the dCas9 principle, CRISPR activation (CRISPRa) can be utilized for transcriptional activation, whereas CRISPR interference (CRISPRi) technology has been created to achieve transcriptional suppression [82,87,88]. For bacteria to retain their native cellular metabolism, transcriptional regulation is necessary [89]. The time needed for metabolic engineering is greatly decreased due to the flexible multiplexed gene silencing provided by the CRISPRi method without the need for DSBs induction. Therefore, CRISPRi offers exceptional benefits for regulating important gene expression, examining undiscovered gene functions, and screening target genes at the genome level [89]. Myrbraten et al. developed a two-plasmid CRISPRi system in which dCas9 and genespecific unidirectional guide RNA (sgRNA) were expressed on different plasmids and could effectively inhibit the expression of any target genes [81]. Using the CRISPRi method to knockdown the phenotype of the genes producing cell wall hydrolase acm2, DNA replication start gene DnaA, and early cell division protein ezrA in Lactobacillus, the function of critical cell cycle genes in L. plantarum was discovered. This study offers the ideal illustration of how to rapidly screen for critical and non-critical genes using CRISPRi.

Future Perspectives
As a microorganism closely tied to human life, Lactobacillus plays a crucial function in agriculture, industry, and medicine. Therefore, it is essential to develop unique and superior Lactobacilli that fulfill human needs. To increase the performance of cell factories in synthetic biology, CRISPR/Cas9-based genome editing technology may perform efficient and selective gene editing in bacteria. However, it is rarely applied to Lactobacillus.

Improvement of Immune Regulation Traits
Most Lactobacilli have potential immune regulation functions, and CRISPR/Cas9-based genome editing can be used to transform Lactobacillus and enhance its immune regulation characteristics. Thomas et al. found out that L. reuteri-derived histamine can inhibit the production of human TNF (a pro-inflammatory cytokine) as an immune protein in terms of mediating cellular inflammation [90]. Through the H2 receptor, histamine activates AC (adenylate cyclase) and increases intracellular cAMP (cyclic adenosine monophosphate) levels, which can decrease TNF synthesis at the transcriptional level. Histamine is produced via histidine decarboxylase-catalyzed decarboxylation of histidine. Therefore, the expression of the amino acid decarboxylase gene cluster is essential for histamine synthesis. If CRISPR/Cas9-based genome editing is utilized to increase the expression of this gene, the immune regulation functions of these strains can be boosted in a stable and durable manner.

Increased Resistance to Environmental Stress
Lactobacilli are typically fermented in a harsh industrial environment in the food sector. Additionally, after being consumed by humans as probiotics, they must compete with the gut microbiota and the highly dynamic gastrointestinal environment. Using CRISPR/Cas9, the genome of Lactobacillus was modified to make it more stable and adaptable to many environmental circumstances.
Lactobacillus produces lactic acid and other organic acids through fermentation, and the accumulated acids will affect their metabolic growth and function. Therefore, Lactobacillus needs to have acid resistance. The glutamate decarboxylase (GAD) system is an important acid tolerant system of Lactobacillus [91]. Gong et al. discovered that the transcriptional regulator GadR has a positive regulatory effect on the system, and that the high expression of the gadR gene can improve L. brevis' acid tolerance [92]. Following the construction of an in-frame glnR deletion strain of L. brevis, it was discovered that the GlnR protein produced by the glnR gene can negatively control the glutamate decarboxylase system and that the deletion of glnR in L. brevis results in increased acid tolerance [93]. Therefore, hyper-expression of the gadR gene or knock-out of the glnR gene using CRISPR/Cas9-based genome editing may be an effective method to enhance the stress resistance of the strain.

Development of Efficient Mucosal Vaccines
The best way to induce innate and adaptive mucosal immune responses is to immunize the mucosa directly, rather than through the systemic route (parenteral injection). Mucosal vaccines can also induce serum antibodies and systemic cell-mediated responses [94]. Due to its ease of administration and mucosal co-immune system, which allows the induction of immune responses on one mucosal surface and then the movement of immune cells to other distant mucosal sites, mucosal delivery is a particularly attractive vaccination strategy [95]. Therefore, there is a need to develop a vector to protect vaccine antigens from the adverse mucosal environment and deliver them to mucosa-associated lymphoid tissues. Lactobacillus, as a safe bacteria that can survive in the human body environment and with the ability to regulate the immune system, can be regarded as a potential vaccine carrier [96].
Trichinellosis is a food-borne parasitic disease that can infect both humans and animals, causing serious economic losses to the animal husbandry and food industry, and also endangering human health. Therefore, the development of a vaccine against Trichinella spiralis infection is crucial [97]. Recombinant L. plantarum containing TsCPF1 antigen and IL-4 (interleukin-4) was constructed by Xue et al. [98]. Oral immunization in mice significantly stimulated the production of anti-TsCPF1-specific IgG antibody, strong mucosal IgA reaction, and Th1/Th2 mixed immune response, thereby reducing intestinal injury significantly.
TsCPF1 is an effective candidate antigen against Trichinella spiralis infection, while IL-4 is an excellent adjuvant against Trichinella spiralis. In addition, invasive L. plantarum carrying TsCPF1 and IL-4 antigens showed the best protective effect and served as a new vaccine against T. spiralis infection.
Therefore, through CRISPR/Cas9-based genome editing, the required antigen and adjuvant were cloned into the plasmid as template donors with the Cas9 complex, and properly inserted into the bacteria, which could be applied to treat disease.

Conclusions and Discussion
As a microorganism closely associated with human life, Lactobacillus plays an essential role in agriculture, industry, medicine, and other fields. It is particularly important to develop some new Lactobacillus strains with excellent performance. Although there are numerous species of Lactobacillus, conventional screening of Lactobacillus is a laborintensive and time-consuming task. In addition, as more and more Lactobacillus genomes are sequenced, a large number of unknown genes must be investigated for their function. However, conventional methods of Lactobacillus gene manipulation are labor-intensive and inefficient, so there is an urgent need to develop fast and efficient genetic manipulation tools. In recent years, the technique of producing superior strains through CRISPR/Cas9based genome editing has captured the interest of a huge number of researchers. Compared with earlier gene editing technologies, CRISPR/Cas9-based genome editing technology has less operational difficulty, low editing cost, and higher targeting efficiency. However, there are few reports of gene editing in Lactobacillus, mainly because the homologous recombination ability of Lactobacillus is limited, and it is difficult to edit multiple targeted genes at the same time. In addition, Lactobacilli are extremely susceptible to death after creating DSBs in several locations. We can, therefore, attempt to use Cas9 nickase for gene editing. Insufficient expression of gRNA will also affect subsequent genome editing, and the expression of gRNA can be increased by replacing strong promoters. Additionally, to reduce the off-target effects of the Cas9 system, the properties of Cas9 protein can be changed to make it more accurate when targeting genes or to increase the targeting effectiveness of Cas9 by creating more suitable PAM sequences. Researchers have also created a series of high-fidelity Cas9 variations to increase the editing fidelity or targeting range of CRISPR/Cas9 system [99]. However, additional experimental verification is needed to confirm their applicability to Lactobacillus. Using two CRISPR/Cas9-based genome editing approaches on three strains of L. plantarum, Leenay et al. discovered that the success rate of editing the same region in different L. plantarum strains using the two methods varied [67]. Therefore, other procedures must be created for multiple strains.
In addition to the external CRISPR/Cas9 system, Lactobacillus also possesses an endogenous CRISPR/Cas system that can be exploited for targeted gene editing by developing an appropriate gRNA. Hidalgo-Cantabrana et al. successfully altered L. crispatus using the endogenous type I-E CRISPR/Cas system, including gene knockout, gene insertion, and various alterations, such as stop codon insertion and single nucleotide replacement [100].
The application of CRISPR/Cas9-based genome editing to Lactobacillus has made some progress thus far. Genome editing using CRISPR/Cas9 to improve Lactobacillus may be a promising method. However, Lactobacillus is extremely diverse, making it challenging to find a universal gene editing tool that is appropriate for all strains. In the future, it will be important to develop more diverse systems for editing the Lactobacillus genome.