Development of an Efficient Gene Insertion Tool in Bacillus subtilis Based on CRISPR-associated transposase systems

Development of an Efficient Gene Insertion Tool in Bacillus subtilis Based on CRISPR-associated transposase systems | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of an Efficient Gene Insertion Tool in Bacillus subtilis Based on CRISPR-associated transposase systems Shumin Chen, Xuyang Zhu, Shengqi Gao, Xinrui Yu, Jiazheng Shen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8770286/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacillus subtilis is a pivotal model organism in both industrial biotechnology and scientific research, where the efficiency of its genetic engineering is very important. However, achieving highly efficient gene insertion in this bacterium remains a significant technical challenge. To address this, we aimed to develop a novel gene insertion tool in B. subtilis . Building upon the Vibrio cholerae -derived Vch CAST system, we systematically optimized and successfully established a high-performance VchCAST system. The core components of this system include the TniQ-Cas678 complex, a guide RNA for precise targeting, and the TnsABC transposase complex responsible for DNA integration. Under antibiotic selection, screening and employing a strong promoter to drive crRNA expression increased the single-locus transposition efficiency to 41%. Subsequent genomic integration of the transposase operational unit further enhanced the efficiency to 80%. Moreover, we demonstrated that overexpressing the auxiliary factor BmrR enables simultaneous integration at two distinct genomic loci. Through protein engineering of the key transposase TnsB, we obtained optimized variants V178F and V178L with significantly enhanced activity, which improved the overall transposition efficiency by 232.6% and 178.07%, respectively. We then conducted transposition validation with the optimized system, achieving a site-specific gene insertion efficiency of approximately 95.25%. In conclusion, this study not only provides a robust gene insertion platform for B. subtilis microbial cell factory engineering, but also stands as a valuable reference for the construction of gene insertion tool in other microbial. Bacillus subtilis Gene insertion CRISPR-associated transposase TnsB Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bacillus subtilis , as a typical Gram-positive bacterium and model industrial microorganism, boasts advantages such as non-pathogenicity, strong protein secretion capability, a clear genetic background, and mature fermentation processes. 1 – 3 Consequently, it is widely used in biosynthesis fields like enzyme preparation, vitamin, and functional sugar. 4 – 7 Therefore, achieving precise and efficient genome editing in this host is of great significance for enhancing the ability of B. subtilis to synthesize target products. Genome integration technology, which involves inserting foreign genes into the host chromosome, not only eliminates the dependence on antibiotic selection, enabling stable inheritance of genetic elements 8 , 9 and ensuring the economic viability and biosafety of industrial fermentation, but also reduces the metabolic burden on the host strain and improves the synthesis level of the target product. 10 – 12 Currently, the most efficient genome integration method is the CRISPR-Cas9 technology. 6 , 13 , 14 However, it causes DNA double-strand breaks and requires the host's homologous directed repair (HDR) system for completion. Since the HDR efficiency in bacterial strains is limited, the efficiency of fragment insertion mediated by CRISPR-Cas9, especially for large fragments, remains low. 15 – 17 Thus, developing efficient gene integration tools for B. subtilis is highly urgently. Transposons are genetic elements capable of moving and inserting into new sites within the genome. 18 , 19 They amplify themselves through "cut-and-paste" or "copy-and-paste" mechanisms but inherently lack programmable targeting capability. In 2019, Samuel H.Sternberg team discovered a Tn7-like transposon system in Vibrio cholerae . 20 During evolution, this system interacts with the CRISPR system, combining the targeting specificity of CRISPR-Cas with the autonomous integration mechanism of transposons, thereby overcoming the non-targeted random insertion issue of traditional transposon insertion technologies and enabling programmable integration independent of homologous recombination. In all 25 test cases, the CRISPR-associated transposase (CAST) system was accurately delivered to the predetermined target sites, demonstrating extremely high specificity. In the same year, Zhang Feng team also identified a CAST system from Scytonema hofmanni and used this system to achieve site-specific integration of a 2.5 kb DNA fragment in Escherichia coli with an insertion efficiency of 80%. 21 These two studies not only expanded the understanding of the functional diversity of CRISPR-Cas systems but also established a new paradigm for the precise insertion of large DNA fragments, laying the foundation for CAST systems as powerful gene insertion tools. Since then, CAST systems from more sources have been continuously discovered and functionally characterized, driving ongoing progress in this field. The VchCAST system derived from V. cholerae is one of the most well-characterized Type I-F CAST systems. Studies have confirmed that it can achieve nearly 100% insertion efficiency in various hosts such as E. coli and Tatumella citrea . 20 , 22 The molecular mechanism of this system is highly coordinated. Its core functional units include the QCascade complex (composed of TniQ, Cas6, Cas7, and Cas8), 23–25 the transposase components (TnsA, TnsB, and TnsC), and the donor plasmid carrying the cargo of interest. A key feature of this donor plasmid is the specific cargo gene of interest flanked by the Right End (RE) and Left End (LE) sequences. These RE and LE sequences serve as recognition and cleavage sites for the transposase, precisely delineating the cargo to be mobilized and determining its orientation (RL direction) upon integration into the target site. The mechanism of action is as follows: the QCascade complex first recognizes and binds to the target DNA, forming an R-loop structure, then recruits the ATPase TnsC and the TnsAB transposase, ultimately precisely inserting the donor DNA fragment from the donor plasmid about 49 bp downstream of the target site. 26 – 29 The entire process does not rely on homologous recombination repair and exhibits a clear RL direction preference due to the asymmetric structure of the transposon ends. Given its theoretical capabilities in gene knockout and multi-copy integration, this system shows great potential in synthetic biology and metabolic engineering, making it an ideal tool for building mutant library and optimizing biosynthetic pathway. However, although the VchCAST system exhibited extremely high editing efficiency in some hosts, its performance in the Gram-positive model strain B. subtilis was significantly poor, with a native efficiency of only 0.00018%. Even with low-temperature cultivation (16°C), the efficiency only increased to 3.64%. This limitation severely restricts its application in this key chassis organism. 30 This study is dedicated to developing a VchCAST-based gene integration tool for B. subtilis . Firstly, through the coordinated optimization of the crRNA expression element and transposase expression, efficient single-site integration was achieved. By investigating accessory factors affecting transposition efficiency, it was found that co-expressing specific accessory factors enabled simultaneously dual-site gene insertion, revealing the potential for multiple genomic integrations. Furthermore, protein engineering of TnsB significantly enhanced its gene integration capability. Ultimately, we substantially improved the integration efficiency of the VchCAST system in B. subtilis , constructing a powerful microbial cell factory engineering platform for this chassis organism and standing valuable reference for other bacteria. Materials and Methods Culture Media and Antibiotics LB medium (Luria-Bertani Medium) was composed of 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl. For solid medium, 2% (w/v) agar was added. YN medium (Yeast Nitrogen Medium), used for preparing competent B. subtilis cells, contained 7 g/L yeast extract and 18 g/L nutrient broth. For selection of different antibiotic resistance markers, the following concentrations were used: ampicillin (Amp) 100 µg/mL, tetracycline (Tet) 25 µg/mL, kanamycin (Kana) 30 µg/mL, chloramphenicol (Cmr) 5 µg/mL, and spectinomycin (Spe) 40 µg/mL. Antibiotics were typically added to media at 0.1% (v/v) of stock solutions, except for spectinomycin selection in B. subtilis , which required 0.2% (v/v) addition. Plasmid Construction All plasmids and bacterial strains used in this study are listed in Table S1 , and oligonucleotide sequences are provided in Table S2. E. coli JM109 and B. subtilis SCK6 were employed as hosts for plasmid construction. The backbone plasmid pAD123 was maintained in our laboratory collection. The TnsABC protein sequences in plasmid pBSTnp, as well as the left-end (LE) and right-end (RE) sequences in plasmid pBSDonor, were synthesized based on sequences reported by Sternberg et al. The B. subtilis strains WS9CPG and WS9CPS, which harbor chromosomally integrated QCascade complexes, were also preserved in our laboratory. Plasmid sequencing and gene synthesis services were provided by Genewiz (Suzhou, China). During the construction of the pBSTnp plasmid, the pAD vector backbone, promoter sequence, and TnsABC protein coding sequence were first amplified by conventional PCR. The promoter and TnsABC sequences were then connected using overlap extension PCR to obtain a TnsABC expression cassette (containing the complete promoter and TnsABC coding region) flanked by 25-bp homologous arms. Subsequently, the expression cassette was ligated with the linearized vector via one-step cloning. A 10-µL aliquot of the ligation product was transformed into chemically competent B. subtilis SCK6 cells. The transformed culture was spread on LB solid plates containing chloramphenicol and incubated overnight at 37°C. Single colonies were picked and inoculated into 10 mL of LB liquid medium for cultivation. The recombinant plasmid was confirmed through plasmid extraction, restriction enzyme digestion verification, and sequencing analysis, ultimately yielding the target engineered strain that met the design requirements. The primers used in this study are listed in Supplementary Table 1, the constructed plasmids and the guide RNAs used are listed in Supplementary Table 2, and the main DNA sequences are listed in Supplementary Table 3. Genomic Integration and Marker Excision This study employed the Cre/lox recombination system to integrate the TnsABC expression cassette into the B. subtilis strain WS9CPS. This strain harbors a green fluorescent protein (GFP) gene pre-integrated at the nprE locus, while the QCascade complex expression cassette was site-specifically integrated at the lacA locus. The TnsABC expression cassette was subsequently integrated at the mpr locus. The integration vector was constructed using pET24a as the backbone. For each integration site, 1000-bp fragments corresponding to the upstream and downstream regions were selected as homologous arms. These homologous arms, along with the integration cassette and a Cre/lox-tagged antibiotic resistance gene, were individually amplified and assembled pairwise via overlap extension PCR. Each adjacent fragment was designed with 20–25 bp homologous sequences. The assembled fragments were then ligated into the linearized vector using one-step isothermal assembly. The successfully constructed integration vectors were transformed into competent WS9CPS cells to generate the integrated strains. To remove the antibiotic resistance markers, a Cre recombinase expression plasmid containing a temperature-sensitive origin of replication was introduced into the integrated strains. Transformants were spread on LB plates supplemented with 0.1 mM IPTG to induce Cre recombinase expression. Subsequently, the transformants were inoculated into both antibiotic-containing and antibiotic-free media and incubated at 50°C for 12 hours, ultimately yielding marker-free integrated strains. CAST Transposition Assay Strains harboring genomically integrated VchCAST expression cassettes were cultured in LB medium for 10 h. Subsequently, 500 µL of the culture was transferred to YN medium supplemented with 250 µL of xylose solution (final concentration: 400 mg/L) for induction. After 4 h of incubation, cells were aliquoted to prepare competent cells. A plasmid encoding crRNA and the cargo gene was introduced via chemical transformation, and transformants were selected on solid LB plates containing tetracycline, followed by incubation at 37℃ for 12 h. Colonies were resuspended in LB, serially diluted (10⁻⁵ to 10⁻⁷), and 100 µL of each dilution was spread on plates. After 16 h of incubation at 37°C, the above steps were repeated. The resuspended cells were diluted appropriately and plated for single-colony isolation, followed by overnight incubation at 51°C. Individual colonies were randomly selected for PCR validation, or genomic DNA was extracted from the pooled cell suspension for qPCR analysis. Quantitative Real-Time PCR (qPCR) Colonies from plates were resuspended in sterile water, and genomic DNA was extracted using the Bacterial Genomic DNA Extraction Kit (Tiangen Biotech, Beijing) according to the manufacturer’s instructions, with final storage at -20°C. QPCR was performed using PerfectStart® Green qPCR SuperMix, with duplicate reactions for each sample. Each 20 uL reaction mixture contained 0.4 µL each of forward and reverse primers, 2 µL of genomic DNA, 10 uL of 2× PerfectStart® Green qPCR SuperMix, and 7.2 µL of water. Reactions were prepared in a 96-well plate and analyzed on a QuantStudio 3 Real-Time PCR System using the -ΔΔCt method. All data in the text and figures come from two or three independent biological replicates. The qPCR primers used in this study are listed in Supplementary Table 1. Measurement of Gene Insertion Efficiency For systems with low editing efficiency or when validating single-locus insertion efficiency of mutants, quantitative PCR (qPCR) was employed for accurate quantification. To ensure experimental rigor in cases of high single-locus efficiency or when assessing dual-locus insertion efficiency, at least 25–50 single colonies were randomly selected from the final plasmid-cured plates for colony PCR analysis. The insertion efficiency was calculated as the percentage of positive clones relative to the total number of colonies tested. The PCR primers used in this study are listed in Supplementary Table 1. Results Construction and Validation of VchCAST System in B. subtilis To develop a programmable gene integration tool in B. subtilis , we engineered V. cholerae -derived VchCAST system that enables precise insertion of DNA cargo into specific genomic loci by leveraging the RNA-guided targeting capability of CRISPR-Cas and the integration mechanism of transposon. 31 The transposition system was developed using the laboratory-preserved host strains WS9CPG and WS9CPS. These strains were previously engineered via chromosomal integration of a Cas678-TniQ containing expression module. The four genes cas678 - tniQ were constructed into two expression cassettes tniQ - cas7 and cas6 – cas8 , each regulated by different constitutive promoters and integrated into the lacA locus of B. subtilis . Within the same expression cassette, the two gene units were linked in tandem by inserting an endogenous B. subtilis RBS sequence (AAGGAGTGTCAAGA). In strains WS9CPG and WS9CPS, the expression cassette containing TniQ and Cas7 proteins are expressed under the control of promoters P gsiB and P sunA , respectively, while the expression cassette containing Cas8 and Cas6 proteins is both driven by the promoter P groES . We subsequently assembled the transposase expression plasmid pBSTnp and donor plasmid pBSDonor (Fig. 1 A).The transposase expression plasmid pBSTnp was generated by cloning genes encoding key transposition proteins (TnsA, TnsB, and TnsC) downstream of a tandem dual-promoter P HpaII -P amyQ' , followed by insertion into a modified pAD vector backbone containing the B. subtilis replication origin and a chloramphenicol resistance marker. Meanwhile, we constructed the donor plasmid pBSDonor using the temperature-sensitive replication vector pE194. This plasmid contains two key functional units: a crRNA expression cassette driven by the P veg promoter (designed to guide the CRISPR-Cas complex to specific genomic loci), and a spectinomycin resistance gene that serves as both the cargo and a selectable marker. The use of a temperature-sensitive replication origin in this donor plasmid facilitates its efficient clearance post-integration, thereby minimizing the likelihood of false-positive colonies during transposition validation. Figure 1 . Development and optimization of a VchCAST-based programmable gene integration system in Bacillus subtilis . (A) Schematic diagrams of the engineered host strains WS9CPG and WS9CPS, along with the transposase and donor plasmids. The pBSTnp plasmid contains the P amyQ' and P HpaII promoters, as well as the tnsA , tnsB , and tnsC genes. The pBSDonor plasmid includes a P veg promoter, a crRNA composed of two repeat sequences (gray diamonds) and a spacer sequence (reddish-brown rectangle), and the RE-cargo ( spe )-LE structure. (B) Illustration of the transposition experimental workflow of the VchCAST system in Bacillus subtilis. (C) Colony PCR validation. Detecting whether the system can achieve gene insertion in Bacillus subtilis strains WS9CPG and WS9CPS after resistance screening. (D) Sanger sequencing chromatograms of the upstream junction of the transposon in the RL integration orientation after performing transposition experiments in strain WS9CPS. The plasmids pBSTnp and pBSDonor were sequentially introduced into WS9CPG and WS9CPS host strains to establish two dual-plasmid editing systems. Following two rounds of subculturing at 37°C to mediate gene insertion, the bacterial cells were subjected to 51°C for 12 h for plasmid elimination (Figure. 1B). Single clones were obtained through resistance screening and verified by PCR amplification and sequencing, confirming precise integration of the cargo gene 49 bp downstream of the crRNA target site (Fig. 1 D). Comparison of system efficiency demonstrated that when the P sunA promoter was used to transcript tniQ - cas7 genes, the resulting transposition efficiency was significantly higher than that of the P gsiB promoter-controlled system (Fig. 1 C). Previous unpublished data from our laboratory using green fluorescent protein (GFP) to characterize promoter activity demonstrated that P sunA possesses stronger transcriptional activity than P gsiB , which we hypothesize to be the key factor enhancing the efficiency of the transposition system. Optimization of crRNA Promoter and TnsABC Expression After optimizing the expression strength of Cas protein and TniQ, we then attempted to improve transposition efficiency by optimizing expression of two core components: crRNA and transposase. Initially, we replaced the original crRNA promoter P veg with six constitutive promoters—P sunA , P citZ , P groESL , P 43 , P HpaII , and P amyQ' —to regulate crRNA expression (Table 1 and Fig. 2 A). Following elimination of the pBSDonor plasmid, genomic DNA was extracted from colonies, and quantitative PCR (qPCR) was performed using primers specific to the genomic target site and the left (LE) and right (RE) ends of the transposon, with the epr gene as an internal reference (Fig. 2 B). Each sample was analyzed by qPCR through three parallel reactions targeting the internal reference gene, the RL integration direction, and the LR integration direction, respectively. The transposition efficiency for each orientation was calculated using the formula 2 −ΔCt , where ΔCt represents the difference between the Ct value of the experimental reaction and that of the internal reference gene. The total transposition efficiency of the system was defined as the sum of the efficiencies for both orientations. Results demonstrated that the P citZ promoter achieved a transposition efficiency of 41% for the VchCAST system, indicating its superior capability in this context (Fig. 2 C). Table 1 We constructed a more stable transposition system by integrating the TnsABC transposase complex into the genome to circumventing plasmid instability. Transposition verification demonstrated that following plasmid clearance and antibiotic selection to reduce false-positive background, the single-plasmid VchCAST system achieved 80% transposition efficiency, significantly outperforming the dual-plasmid configuration (Fig. 2 D). Figure 2 . Optimization of the VchCAST System. (A) Schematic diagram of promoter optimization in the VchCAST system. The original promoter P veg , responsible for regulating crRNA expression, was replaced with various alternative promoters. (B) Schematic illustration of the two possible integration orientations (RL and LR), along with the design of specific qPCR primer pairs used for their selective detection. (C) Evaluation of the influence of different promoters on transposition efficiency by qPCR. Data are presented as mean ± SD of n = 3 independent biological replicates. (D) Colony PCR verification. Detection of gene insertion efficiency of the VchCAST system after integration of the TnsABC expression cassette into the genome. Considering the large size of the TnsABC complex, we further tested a split-expression strategy by distributing TnsA, TnsB, and TnsC into two independent expression cassettes (Supplementary Figure S1 A). TnsB, the largest and most critical subunit for DNA binding, was expressed under the P groES promoter, while TnsA and TnsC were co-expressed from the original dual-promoter P HpaII -P amyQ' , with an endogenous B. subtilis RBS sequence inserted between them. Contrary to expectations, this partitioned expression markedly reduced transposition efficiency (Supplementary Figure S1 B), suggesting that the single expression cassette structure of the TnsABC complex may be more favorable for its proper expression. 32 Dual-site Transposition of VchCAST System in B. subtilis A notable advantage of the VchCAST transposon system lies in its capacity for simultaneous integration in multiple genome sites. 22 , 33 This "one-step" integration strategy effectively circumvents the traditional sequential genetic manipulations, thereby substantially shortening the timeline for strain construction and optimization. Having established the system's high efficiency in single-site insertion, we further investigated its capability for simultaneously integration at dual distinct genomic loci. Figure 3 . Optimization of the VchCAST system for multi dual-site gene insertion. (A) Schematic of the donor plasmid designed for dual-site gene insertion. The original single-site crRNA has been replaced with a targeting RNA directed against the epr and nprB loci. Additionally, the resistance gene is flanked by lox71 and lox66 sites, enabling subsequent Cre recombinase-mediated marker excision and thus supporting iterative rounds of integration. (B) The effect of overexpressing different host accessory factors on the transposition efficiency of the system was validated by colony PCR. The bar graph illustrates the single-site and dual-site insertion efficiencies for each experimental group. Insertion efficiency was calculated as the ratio of the number of positive clones to the total number of picked colonies in each experimental group. As the insertion efficiency was derived from a single round of colony PCR-based qualitative analysis, where only one set of colony picking and identification was performed per condition without experimental replicates, no error bars are shown in the graph. We selected two genomic sites ( epr and nprB ) and constructed a dual-target donor plasmid. Furthermore, the spectinomycin resistance gene on the donor plasmid was flanked by lox71 and lox66 sites, which can be recognized by Cre recombinase. This design enables, upon successful gene integration, the removal of the spe resistance marker via the Cre- lox system, thereby allowing subsequent rounds of transformation with new donor plasmids for stacking additional insertion sites (Fig. 3 A). However, during the initial assessment of dual-site transposition efficiency, while multiple clones with single-site insertions were obtained, no strains exhibiting simultaneous integration at both loci were identified. This outcome may indicate that under current conditions, the system's efficiency remains insufficient to support robust dual-site gene integration. To enhance the efficiency of dual-site simultaneous insertion, this study systematically screened host factors that may positively regulate the transposition process. Among these, the DNA-binding protein IhfA/B, as an integration host factor, can induce DNA conformational changes and influence recombination processes; 34 the HU protein subunits HupA and HupB have been reported to play a role in phage transposition; 35, 36 the transcriptional regulator BmrR, belonging to the MerR family, may indirectly modulate the expression of genes associated with transposition system function by inducing conformational changes in the DNA of target gene promoter regions; 37 the DNA-binding and -bending protein Fis is frequently involved in site-specific recombination and transcriptional regulation; 38 and the ATP-dependent protease ClpX may influence the dissociation of post-integration transposition complex. 33 Based on this background, we selected these six potentially relevant factors for investigation. By constructing their respective overexpression systems, followed by two rounds of passage culture and antibiotic screening, the transposition efficiency was systematically analyzed using colony PCR combined with nucleic acid gel electrophoresis. The results indicated that among the 24 picked monoclonal colonies, strains with single-site insertions were observed for all six factors, with varying numbers of insertions (Fig. 3 B and Supplementary Figure S2). In the Fis overexpression group, only one colony exhibited dual-site gene insertion, while the HupB overexpression group yielded five colonies with dual-site insertions. However, no colonies with simultaneous dual-site insertion were obtained in the IhfA/B, ClpX, or HupA overexpression groups, possibly due to the relatively low transposition efficiency of the system when these factors are overexpressed. Notably, BmrR overexpression demonstrated the highest dual-targeting efficiency: six out of 25 monoclonal colonies successfully achieved simultaneous integration at both the epr and nprB genomic sites, indicating its significant role in enhancing dual-site integration capability. Site-Directed Mutagenesis of Transposase TnsB During natural evolution, the wild-type CRISPR-associated transposase (CAST) system, shaped by the survival needs of the host strain, does not exhibit the highest transposition efficiency. Excessively high transposition efficiency may impose an additional burden on the strain. 39 Therefore, the CAST system likely balances resource competition and survival pressure while maintaining essential functions and host viability, thereby achieving evolutionary stability. To overcome this limitation in the transposition capability of the wild-type system, we employed a semi-rational design strategy to engineer the CAST system with the aim of enhancing its transposition efficiency. As the key catalytic enzyme in the transposition process, TnsB is responsible for specifically recognizing transposon terminal sequences, cleaving target DNA strands, and mediating the connection between transposons and target sites. Its catalytic efficiency directly influence the overall integration capacity of the system. In this study, we implemented a computer-aided semi-rational protein design strategy to engineer TnsB. To systematically identify potential optimization sites, we integrated the PROSS algorithm 40 and HotSpot Wizard tool 41 to perform multi-scale analysis and beneficial mutation anticipating of the TnsB protein. Based on computer anticipation and structure analysis, we selected 17 candidate sites for subsequent experimental validation. Figure 4 . Engineering TnsB variants to obtain an efficient gene integration transposase system. (A) Effect of TnsB mutants on the gene insertion efficiency of the system. The efficiency of gene insertion by the transposon system was detected by qPCR, and the optimized enhancement efficiency was calculated by taking the ratio of the experimental group to the control group and then comparing it to the control group. (B) Multiple sequence alignment analysis of TnsB protein. (C) Effect of a site-directed mutation at amino acid position 178 of TnsB on the system's gene insertion efficiency. (D) Evaluation of system gene insertion efficiency after integrating the dominant mutants into the genome. (E) Structural prediction and simulation of the Vch TnsAB complex. Structure of the Vch TnsAB complex predicted by AlphaFold3 (center), further simulated in a conformation bound to DNA. Recently resolved crystal structure of the Pse TnsAB complex (left) is shown for comparison. 39 In the figure, different monomers are represented by different colors: TnsB tetramers are shown in dark green, blue, purple, and light green representing TnsB.1 to TnsB.4; TnsA tetramers are shown in gray, yellow, pink, and cyan representing TnsA.1 to TnsA.4. (Right) A close-up shows the mutated V178 residue (red), whose predicted position is adjacent to transposon DNA (orange), suggesting it may participate in DNA interaction. Data in (A) (C) (D) are presented as mean ± SD of n = 3 independent biological replicates. Using a dual-plasmid expression system consisting of the pBSTnp plasmid carrying TnsABC transposase and the pBSDonor plasmid carrying the target gene as the wild-type control platform, we constructed seventeen TnsB mutants (S28D, C31D, A70P, V178F, G194K, S215Y, L216A, V216L, V227L, E290P, L304H, L326G, D356T, K399N, E421P, E424P, M493K) and evaluated their transposition efficiency. The transposition efficiency of each mutant was systematically assessed through antibiotic resistance screening and real-time quantitative PCR analysis. The experimental results showed that among the 17 candidate sites, eight mutants (C31D, V178F, V227L, E290P, L304H, M493K, E424P, and L326G) exhibited improved transposition efficiency, with the V178F mutant demonstrating the most significant enhancement—an increase of 232.6% (Fig. 4 A). Subsequently, we performed sequence alignment analysis on the 178 site of TnsB (Fig. 4 B and Supplementary Figure S3). Based on the alignment results, this site was mutated to leucine (L), isoleucine (I), and arginine (R), and the functional effects of these mutants were validated through transposition assays. qPCR results showed that when the 178 site was mutated to phenylalanine (F), the system maintained the highest transposition efficiency, while the V178L mutant also demonstrated a substantial improvement, with its relative transposition efficiency increasing by 178.07% (Fig. 4 C). To further verify the enhanced gene insertion efficiency of the screened TnsB mutants in the transposition system, we integrated the TnsABC expression cassettes carrying the two advantageous mutants, V178F and V178L, into the genome. After subculturing and antibiotic screening, qPCR analysis (Fig. 4 D) indicated that the transposition efficiencies of the V178F and V178L systems were 95.25% and 90.24%, respectively. Additionally, we selected 24 single clones for colony PCR validation. Agarose gel electrophoresis results showed that the system mediated by the V178F mutant yielded 24 positive clones, while the V178L-mediated system yielded 22 positive clones, both exceeding the control group (Supplementary Figure S3). Finally, we utilized AlphaFold3 to simulate the structure of the TnsAB protein and its bound DNA, and mapped the corresponding mutant residues of TnsB onto the predicted structure of the TnsAB tetramer (Fig. 4 E). Structural analysis revealed that this residue is located near the DNA strand, indicating that the mutation at this site may enhance integration efficiency by modulating protein-DNA interactions. Discussion The VchCAST transposase system originating from V. cholerae represents an emerging genome editing technology capable of precise integration of large DNA fragments without relying on DNA double-strand breaks and homologous recombination repair. This study developed a VchCAST-based gene integration tool suitable for B. subtilis . Initially, we successfully established the type I-F VchCAST system from V. cholerae in B. subtilis . The system efficiency demonstrated strong correlation with the expression levels of core components, particularly evidenced by the enhanced transposition efficiency observed when the tniQ - cas7 gene were driven by the strong promoter P sunA . This finding underscores the importance of maintaining high expression levels of key components when reconstructing complex protein machinery in heterologous hosts. Through optimization of the crRNA promoter to P citZ , the system efficiency was elevated to 41%, and genomic integration of the TnsABC transposase operon further enhanced efficiency to 80%, likely attributable to gene integration being more stable than plasmid expression. A key finding was that a split-expression strategy for TnsABC led to a marked decrease in efficiency, demonstrating that co-expression of the three core components within a single operon is more favorable for the expression of the transposase complex. Furthermore, through overexpression of transposition accessory factors, we confirmed the potential of the VchCAST system for simultaneous dual-site integration in B. subtilis . Previous research has demonstrated that BmrR can bind effector molecules and insert between the key promoter regions (-35 and − 10 boxes), inducing bending of double-stranded DNA. This structural modification enables proper recognition by RNA polymerase, thereby indirectly facilitating transcriptional activation. 42 We therefore hypothesize that BmrR's potent DNA-distorting capability may be related to the gene integration process mediated by transposable elements, enhancing the accessibility of multiple target sites to the transposase complex and consequently alleviating efficiency bottlenecks during multiple integration events. This discovery not only provides new perspectives for understanding transposon-host interactions but also establishes novel pathways for enhancing CAST system performance through host environment engineering. Finally, through computer-aided design, we successfully enhanced the transposition performance of TnsB and obtained two potentially optimized variants, V178F and V178L. Based on these findings, subsequent multi-site transposition validation experiments will employ V178F as the preferred engineered variant to evaluate its integration efficiency in industrial settings, thereby providing theoretical foundations and technical support for the broader application of this transposition system in synthetic biology. This study demonstrates that through systematic engineering, the VchCAST-based system enables efficient genome editing in Gram-positive bacteria B. subtilis , significantly expanding the host range of this technology and highlighting the potential of VchCAST as a high-efficiency multi-copy integration tool. Currently, we are actively advancing the practical application of this system in the field of biosynthesis. Future work will focus on developing a VchCAST gene integration tool that operates without antibiotic selection, supports multi-site gene insertion, and is suitable for industrial-scale applications. The preliminary direction involves optimizing transposase activity through rational protein design and modulating its interaction network with host endogenous factors, thereby systematically enhancing transposition efficiency. The ultimate goal is to enable direct screening of positive clones from mixed microbial populations with minimal passaging, completely eliminating reliance on antibiotic-dependent selection methods. Declarations Conflicts of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work received financial support from the National Natural Science Foundation of China (32430081, 32272264, 22478155), the Natural Science Foundation of Jiangsu Province (BE2023686), the National Key Research and Development Program of China (2024YFF1106300), the Fundamental Research Funds for the Central Universities (JUSRP122012), Shenzhen Institute of Synthetic Biology Scientific Research Program (DWKF20210004). Author Contribution SC: Conceptualization, methodology, investigation, formal analysis, and writing the original draft. XZ: methodology, formal analysis, and writing review & editing. SG: methodology, formal analysis, and writing review & editing. XY: methodology. JS: formal analysis and writing the original draft. YC: Visualization. YL: Visualization. HL: Visualization. JW: methodology, writing review, and funding acquisition. LS: Methodology, supervision, and project administration. 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Nat Commun. 2020;11(1):6284. 10.1038/s41467-020-20134-y . Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files floatimage2.png Table 1. Properties of promoters used·for·crRNA optimization. Onlinefloatimage6.png TOC Supplementarymaterials.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8770286","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588336388,"identity":"3fdd8f48-0574-4f37-8560-84106b561c8d","order_by":0,"name":"Shumin Chen","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Shumin","middleName":"","lastName":"Chen","suffix":""},{"id":588336390,"identity":"8e889d15-e2db-4c04-b9d9-e970117d30c1","order_by":1,"name":"Xuyang Zhu","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Xuyang","middleName":"","lastName":"Zhu","suffix":""},{"id":588336392,"identity":"e8714e7c-36f8-4c0a-8d72-01806c1202c7","order_by":2,"name":"Shengqi Gao","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Shengqi","middleName":"","lastName":"Gao","suffix":""},{"id":588336394,"identity":"6e5c6c2e-6890-4709-aa3b-d90bc0c7c1b2","order_by":3,"name":"Xinrui Yu","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Xinrui","middleName":"","lastName":"Yu","suffix":""},{"id":588336397,"identity":"8293d7b6-7f11-4f49-89e8-fb6a1a788068","order_by":4,"name":"Jiazheng Shen","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jiazheng","middleName":"","lastName":"Shen","suffix":""},{"id":588336398,"identity":"a15b6a8c-dcf9-46e3-b79b-49757778ad8d","order_by":5,"name":"Yangyang Chen","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Yangyang","middleName":"","lastName":"Chen","suffix":""},{"id":588336399,"identity":"ee6a7e2b-8504-48a2-8266-3ac15d9d9508","order_by":6,"name":"Yuting Liu","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Liu","suffix":""},{"id":588336404,"identity":"e44754c0-7989-41ea-98c4-eb396390cf56","order_by":7,"name":"Huihui Lv","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Huihui","middleName":"","lastName":"Lv","suffix":""},{"id":588336408,"identity":"131991f5-9a32-4774-988d-9ca14e9fa044","order_by":8,"name":"Jing Wu","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wu","suffix":""},{"id":588336410,"identity":"8a6df147-e17a-4ead-a81e-2d25b747d6b0","order_by":9,"name":"Lingqia Su","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Lingqia","middleName":"","lastName":"Su","suffix":""},{"id":588336412,"identity":"702b213b-9317-4ef2-99bc-0d5ba974d7dc","order_by":10,"name":"Kang Zhang","email":"data:image/png;base64,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","orcid":"","institution":"Jiangnan University","correspondingAuthor":true,"prefix":"","firstName":"Kang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-02-03 03:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8770286/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8770286/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102336108,"identity":"5e2b6ea0-68a1-4682-b417-814530ee28bf","added_by":"auto","created_at":"2026-02-10 16:07:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147834,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment and optimization of a VchCAST-based programmable gene integration system in \u003cem\u003eBacillus subtilis\u003c/em\u003e. (A) Schematic diagrams of the engineered host strains WS9CPG and WS9CPS, along with the transposase and donor plasmids. The pBSTnp plasmid contains the P\u003csub\u003e\u003cem\u003eamyQ'\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003eHpaII\u003c/em\u003e\u003c/sub\u003e promoters, as well as the \u003cem\u003etnsA\u003c/em\u003e, \u003cem\u003etnsB\u003c/em\u003e, and \u003cem\u003etnsC\u003c/em\u003e genes. The pBSDonor plasmid includes a P\u003csub\u003e\u003cem\u003eveg\u003c/em\u003e\u003c/sub\u003e promoter, a crRNA composed of two repeat sequences (gray diamonds) and a spacer sequence (reddish-brown rectangle), and the RE-cargo (\u003cem\u003espe\u003c/em\u003e)-LE structure. (B) Illustration of the transposition experimental workflow of the VchCAST system in Bacillus subtilis. (C) Colony PCR validation. Detecting whether the system can achieve gene insertion in Bacillus subtilis strains WS9CPG and WS9CPS after resistance screening. (D) Sanger sequencing chromatograms of the upstream junction of the transposon in the RL integration orientation after performing transposition experiments in strain WS9CPS.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8770286/v1/aab3659625a4bd539ef94569.png"},{"id":102398241,"identity":"8757beb0-89c5-4875-9c33-2446135bdf96","added_by":"auto","created_at":"2026-02-11 10:21:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76088,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of the VchCAST System. (A) Schematic diagram of promoter optimization in the VchCAST system. The original promoter P\u003csub\u003e\u003cem\u003eveg\u003c/em\u003e\u003c/sub\u003e, responsible for regulating crRNA expression, was replaced with various alternative promoters. (B) Schematic illustration of the two possible integration orientations (RL and LR), along with the design of specific qPCR primer pairs used for their selective detection. (C) Evaluation of the influence of different promoters on transposition efficiency by qPCR. Data are presented as mean ± SD of \u003cem\u003en\u003c/em\u003e=3 independent biological replicates. (D) Colony PCR verification. Detection of gene insertion efficiency of the VchCAST system after integration of the TnsABC expression cassette into the genome.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8770286/v1/1b2386b6b66204b0d782c442.png"},{"id":102397754,"identity":"2e430d8f-4b00-4edd-b4df-4cd54c5bf813","added_by":"auto","created_at":"2026-02-11 10:19:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":79279,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of the VchCAST system for multi dual‑site gene insertion. (A) Schematic of the donor plasmid designed for dual-site gene insertion. The original single-site crRNA has been replaced with a targeting RNA directed against the \u003cem\u003eepr\u003c/em\u003e and \u003cem\u003enprB\u003c/em\u003e loci. Additionally, the resistance gene is flanked by \u003cem\u003elox71\u003c/em\u003e and \u003cem\u003elox66\u003c/em\u003e sites, enabling subsequent Cre recombinase-mediated marker excision and thus supporting iterative rounds of integration. (B) The effect of overexpressing different host accessory factors on the transposition efficiency of the system was validated by colony PCR. The bar graph illustrates the single‑site and dual‑site insertion efficiencies for each experimental group. Insertion efficiency was calculated as the ratio of the number of positive clones to the total number of picked colonies in each experimental group. As the insertion efficiency was derived from a single round of colony PCR-based qualitative analysis, where only one set of colony picking and identification was performed per condition without experimental replicates, no error bars are shown in the graph.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8770286/v1/f6b1ef0680ffe8de85b3ac9c.png"},{"id":102336113,"identity":"5346a2bf-6560-42f4-8b37-1ae4b7f865f7","added_by":"auto","created_at":"2026-02-10 16:07:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":296439,"visible":true,"origin":"","legend":"\u003cp\u003eEngineering TnsB variants to obtain an efficient gene integration transposase system. (A) Effect of TnsB mutants on the gene insertion efficiency of the system. The efficiency of gene insertion by the transposon system was detected by qPCR, and the optimized enhancement efficiency was calculated by taking the ratio of the experimental group to the control group and then comparing it to the control group. (B) Multiple sequence alignment analysis of TnsB protein. (C) Effect of a site-directed mutation at amino acid position 178 of TnsB on the system's gene insertion efficiency. (D) Evaluation of system gene insertion efficiency after integrating the dominant mutants into the genome. (E) Structural prediction and simulation of the \u003cem\u003eVch\u003c/em\u003eTnsAB complex. Structure of the \u003cem\u003eVch\u003c/em\u003eTnsAB complex predicted by AlphaFold3 (center), further simulated in a conformation bound to DNA. Recently resolved crystal structure of the \u003cem\u003ePse\u003c/em\u003eTnsAB complex (left) is shown for comparison.\u003csup\u003e39\u003c/sup\u003e In the figure, different monomers are represented by different colors: TnsB tetramers are shown in dark green, blue, purple, and light green representing TnsB.1 to TnsB.4; TnsA tetramers are shown in gray, yellow, pink, and cyan representing TnsA.1 to TnsA.4. (Right) A close-up shows the mutated V178 residue (red), whose predicted position is adjacent to transposon DNA (orange), suggesting it may participate in DNA interaction. Data in (A) (C) (D) are presented as mean ± SD of \u003cem\u003en\u003c/em\u003e=3 independent biological replicates.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8770286/v1/ef3ad81a2d244eca0d9ed8e3.png"},{"id":104398306,"identity":"3d20c0ee-d29a-40e9-8167-fd6206b91552","added_by":"auto","created_at":"2026-03-11 12:01:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1517285,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8770286/v1/170ba3c9-5fb3-4c6f-a896-8ae5cc3aaa04.pdf"},{"id":102336110,"identity":"d3aa3da7-0492-41ed-91fc-c273b51a8ebe","added_by":"auto","created_at":"2026-02-10 16:07:53","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":45288,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1. Properties of promoters used·for·crRNA optimization.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8770286/v1/cb0b0cec4ac37aa8a36ba2b2.png"},{"id":102962241,"identity":"4dd28d04-1cb3-41ca-bb95-9a8546aea8da","added_by":"auto","created_at":"2026-02-19 04:05:58","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTOC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8770286/v1/00329cbd48ad2787d84eb6db.png"},{"id":102336115,"identity":"f177d3ee-11fa-4f93-b4bd-6f501ea70110","added_by":"auto","created_at":"2026-02-10 16:07:53","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10236282,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.zip","url":"https://assets-eu.researchsquare.com/files/rs-8770286/v1/4f64b758265ebd40e4bf4c48.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of an Efficient Gene Insertion Tool in Bacillus subtilis Based on CRISPR-associated transposase systems","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eBacillus subtilis\u003c/em\u003e, as a typical Gram-positive bacterium and model industrial microorganism, boasts advantages such as non-pathogenicity, strong protein secretion capability, a clear genetic background, and mature fermentation processes.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Consequently, it is widely used in biosynthesis fields like enzyme preparation, vitamin, and functional sugar.\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Therefore, achieving precise and efficient genome editing in this host is of great significance for enhancing the ability of \u003cem\u003eB. subtilis\u003c/em\u003e to synthesize target products.\u003c/p\u003e \u003cp\u003eGenome integration technology, which involves inserting foreign genes into the host chromosome, not only eliminates the dependence on antibiotic selection, enabling stable inheritance of genetic elements\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and ensuring the economic viability and biosafety of industrial fermentation, but also reduces the metabolic burden on the host strain and improves the synthesis level of the target product.\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Currently, the most efficient genome integration method is the CRISPR-Cas9 technology.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e However, it causes DNA double-strand breaks and requires the host's homologous directed repair (HDR) system for completion. Since the HDR efficiency in bacterial strains is limited, the efficiency of fragment insertion mediated by CRISPR-Cas9, especially for large fragments, remains low.\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Thus, developing efficient gene integration tools for \u003cem\u003eB. subtilis\u003c/em\u003e is highly urgently.\u003c/p\u003e \u003cp\u003eTransposons are genetic elements capable of moving and inserting into new sites within the genome.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e They amplify themselves through \"cut-and-paste\" or \"copy-and-paste\" mechanisms but inherently lack programmable targeting capability. In 2019, Samuel H.Sternberg team discovered a Tn7-like transposon system in \u003cem\u003eVibrio cholerae\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e During evolution, this system interacts with the CRISPR system, combining the targeting specificity of CRISPR-Cas with the autonomous integration mechanism of transposons, thereby overcoming the non-targeted random insertion issue of traditional transposon insertion technologies and enabling programmable integration independent of homologous recombination. In all 25 test cases, the CRISPR-associated transposase (CAST) system was accurately delivered to the predetermined target sites, demonstrating extremely high specificity. In the same year, Zhang Feng team also identified a CAST system from \u003cem\u003eScytonema hofmanni\u003c/em\u003e and used this system to achieve site-specific integration of a 2.5 kb DNA fragment in \u003cem\u003eEscherichia coli\u003c/em\u003e with an insertion efficiency of 80%.\u003csup\u003e21\u003c/sup\u003e These two studies not only expanded the understanding of the functional diversity of CRISPR-Cas systems but also established a new paradigm for the precise insertion of large DNA fragments, laying the foundation for CAST systems as powerful gene insertion tools. Since then, CAST systems from more sources have been continuously discovered and functionally characterized, driving ongoing progress in this field.\u003c/p\u003e \u003cp\u003eThe VchCAST system derived from \u003cem\u003eV. cholerae\u003c/em\u003e is one of the most well-characterized Type I-F CAST systems. Studies have confirmed that it can achieve nearly 100% insertion efficiency in various hosts such as \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eTatumella citrea\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The molecular mechanism of this system is highly coordinated. Its core functional units include the QCascade complex (composed of TniQ, Cas6, Cas7, and Cas8),\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e the transposase components (TnsA, TnsB, and TnsC), and the donor plasmid carrying the cargo of interest. A key feature of this donor plasmid is the specific cargo gene of interest flanked by the Right End (RE) and Left End (LE) sequences. These RE and LE sequences serve as recognition and cleavage sites for the transposase, precisely delineating the cargo to be mobilized and determining its orientation (RL direction) upon integration into the target site. The mechanism of action is as follows: the QCascade complex first recognizes and binds to the target DNA, forming an R-loop structure, then recruits the ATPase TnsC and the TnsAB transposase, ultimately precisely inserting the donor DNA fragment from the donor plasmid about 49 bp downstream of the target site.\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e The entire process does not rely on homologous recombination repair and exhibits a clear RL direction preference due to the asymmetric structure of the transposon ends. Given its theoretical capabilities in gene knockout and multi-copy integration, this system shows great potential in synthetic biology and metabolic engineering, making it an ideal tool for building mutant library and optimizing biosynthetic pathway. However, although the VchCAST system exhibited extremely high editing efficiency in some hosts, its performance in the Gram-positive model strain \u003cem\u003eB. subtilis\u003c/em\u003e was significantly poor, with a native efficiency of only 0.00018%. Even with low-temperature cultivation (16\u0026deg;C), the efficiency only increased to 3.64%. This limitation severely restricts its application in this key chassis organism.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis study is dedicated to developing a VchCAST-based gene integration tool for \u003cem\u003eB. subtilis\u003c/em\u003e. Firstly, through the coordinated optimization of the crRNA expression element and transposase expression, efficient single-site integration was achieved. By investigating accessory factors affecting transposition efficiency, it was found that co-expressing specific accessory factors enabled simultaneously dual-site gene insertion, revealing the potential for multiple genomic integrations. Furthermore, protein engineering of TnsB significantly enhanced its gene integration capability. Ultimately, we substantially improved the integration efficiency of the VchCAST system in \u003cem\u003eB. subtilis\u003c/em\u003e, constructing a powerful microbial cell factory engineering platform for this chassis organism and standing valuable reference for other bacteria.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCulture Media and Antibiotics\u003c/h2\u003e \u003cp\u003eLB medium (Luria-Bertani Medium) was composed of 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl. For solid medium, 2% (w/v) agar was added. YN medium (Yeast Nitrogen Medium), used for preparing competent \u003cem\u003eB. subtilis\u003c/em\u003e cells, contained 7 g/L yeast extract and 18 g/L nutrient broth. For selection of different antibiotic resistance markers, the following concentrations were used: ampicillin (Amp) 100 \u0026micro;g/mL, tetracycline (Tet) 25 \u0026micro;g/mL, kanamycin (Kana) 30 \u0026micro;g/mL, chloramphenicol (Cmr) 5 \u0026micro;g/mL, and spectinomycin (Spe) 40 \u0026micro;g/mL. Antibiotics were typically added to media at 0.1% (v/v) of stock solutions, except for spectinomycin selection in \u003cem\u003eB. subtilis\u003c/em\u003e, which required 0.2% (v/v) addition.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid Construction\u003c/h3\u003e\n\u003cp\u003eAll plasmids and bacterial strains used in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and oligonucleotide sequences are provided in Table S2. \u003cem\u003eE. coli\u003c/em\u003e JM109 and \u003cem\u003eB. subtilis\u003c/em\u003e SCK6 were employed as hosts for plasmid construction. The backbone plasmid pAD123 was maintained in our laboratory collection. The TnsABC protein sequences in plasmid pBSTnp, as well as the left-end (LE) and right-end (RE) sequences in plasmid pBSDonor, were synthesized based on sequences reported by Sternberg et al. The \u003cem\u003eB. subtilis\u003c/em\u003e strains WS9CPG and WS9CPS, which harbor chromosomally integrated QCascade complexes, were also preserved in our laboratory. Plasmid sequencing and gene synthesis services were provided by Genewiz (Suzhou, China).\u003c/p\u003e \u003cp\u003eDuring the construction of the pBSTnp plasmid, the pAD vector backbone, promoter sequence, and TnsABC protein coding sequence were first amplified by conventional PCR. The promoter and TnsABC sequences were then connected using overlap extension PCR to obtain a TnsABC expression cassette (containing the complete promoter and TnsABC coding region) flanked by 25-bp homologous arms. Subsequently, the expression cassette was ligated with the linearized vector via one-step cloning. A 10-\u0026micro;L aliquot of the ligation product was transformed into chemically competent \u003cem\u003eB. subtilis\u003c/em\u003e SCK6 cells. The transformed culture was spread on LB solid plates containing chloramphenicol and incubated overnight at 37\u0026deg;C. Single colonies were picked and inoculated into 10 mL of LB liquid medium for cultivation. The recombinant plasmid was confirmed through plasmid extraction, restriction enzyme digestion verification, and sequencing analysis, ultimately yielding the target engineered strain that met the design requirements.\u003c/p\u003e \u003cp\u003eThe primers used in this study are listed in Supplementary Table\u0026nbsp;1, the constructed plasmids and the guide RNAs used are listed in Supplementary Table\u0026nbsp;2, and the main DNA sequences are listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e\n\u003ch3\u003eGenomic Integration and Marker Excision\u003c/h3\u003e\n\u003cp\u003eThis study employed the Cre/lox recombination system to integrate the TnsABC expression cassette into the \u003cem\u003eB. subtilis\u003c/em\u003e strain WS9CPS. This strain harbors a green fluorescent protein (GFP) gene pre-integrated at the \u003cem\u003enprE\u003c/em\u003e locus, while the QCascade complex expression cassette was site-specifically integrated at the \u003cem\u003elacA\u003c/em\u003e locus. The TnsABC expression cassette was subsequently integrated at the \u003cem\u003empr\u003c/em\u003e locus.\u003c/p\u003e \u003cp\u003eThe integration vector was constructed using pET24a as the backbone. For each integration site, 1000-bp fragments corresponding to the upstream and downstream regions were selected as homologous arms. These homologous arms, along with the integration cassette and a Cre/lox-tagged antibiotic resistance gene, were individually amplified and assembled pairwise via overlap extension PCR. Each adjacent fragment was designed with 20\u0026ndash;25 bp homologous sequences. The assembled fragments were then ligated into the linearized vector using one-step isothermal assembly. The successfully constructed integration vectors were transformed into competent WS9CPS cells to generate the integrated strains. To remove the antibiotic resistance markers, a Cre recombinase expression plasmid containing a temperature-sensitive origin of replication was introduced into the integrated strains. Transformants were spread on LB plates supplemented with 0.1 mM IPTG to induce Cre recombinase expression. Subsequently, the transformants were inoculated into both antibiotic-containing and antibiotic-free media and incubated at 50\u0026deg;C for 12 hours, ultimately yielding marker-free integrated strains.\u003c/p\u003e\n\u003ch3\u003eCAST Transposition Assay\u003c/h3\u003e\n\u003cp\u003eStrains harboring genomically integrated VchCAST expression cassettes were cultured in LB medium for 10 h. Subsequently, 500 \u0026micro;L of the culture was transferred to YN medium supplemented with 250 \u0026micro;L of xylose solution (final concentration: 400 mg/L) for induction. After 4 h of incubation, cells were aliquoted to prepare competent cells. A plasmid encoding crRNA and the cargo gene was introduced via chemical transformation, and transformants were selected on solid LB plates containing tetracycline, followed by incubation at 37℃ for 12 h. Colonies were resuspended in LB, serially diluted (10⁻⁵ to 10⁻⁷), and 100 \u0026micro;L of each dilution was spread on plates. After 16 h of incubation at 37\u0026deg;C, the above steps were repeated. The resuspended cells were diluted appropriately and plated for single-colony isolation, followed by overnight incubation at 51\u0026deg;C. Individual colonies were randomly selected for PCR validation, or genomic DNA was extracted from the pooled cell suspension for qPCR analysis.\u003c/p\u003e\n\u003ch3\u003eQuantitative Real-Time PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eColonies from plates were resuspended in sterile water, and genomic DNA was extracted using the Bacterial Genomic DNA Extraction Kit (Tiangen Biotech, Beijing) according to the manufacturer\u0026rsquo;s instructions, with final storage at -20\u0026deg;C. QPCR was performed using PerfectStart\u0026reg; Green qPCR SuperMix, with duplicate reactions for each sample. Each 20 uL reaction mixture contained 0.4 \u0026micro;L each of forward and reverse primers, 2 \u0026micro;L of genomic DNA, 10 uL of 2\u0026times; PerfectStart\u0026reg; Green qPCR SuperMix, and 7.2 \u0026micro;L of water. Reactions were prepared in a 96-well plate and analyzed on a QuantStudio 3 Real-Time PCR System using the -ΔΔCt method. All data in the text and figures come from two or three independent biological replicates.\u003c/p\u003e \u003cp\u003eThe qPCR primers used in this study are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Gene Insertion Efficiency\u003c/h2\u003e \u003cp\u003eFor systems with low editing efficiency or when validating single-locus insertion efficiency of mutants, quantitative PCR (qPCR) was employed for accurate quantification. To ensure experimental rigor in cases of high single-locus efficiency or when assessing dual-locus insertion efficiency, at least 25\u0026ndash;50 single colonies were randomly selected from the final plasmid-cured plates for colony PCR analysis. The insertion efficiency was calculated as the percentage of positive clones relative to the total number of colonies tested.\u003c/p\u003e \u003cp\u003eThe PCR primers used in this study are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eConstruction and Validation of VchCAST System in\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo develop a programmable gene integration tool in \u003cem\u003eB. subtilis\u003c/em\u003e, we engineered \u003cem\u003eV. cholerae\u003c/em\u003e-derived VchCAST system that enables precise insertion of DNA cargo into specific genomic loci by leveraging the RNA-guided targeting capability of CRISPR-Cas and the integration mechanism of transposon.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e The transposition system was developed using the laboratory-preserved host strains WS9CPG and WS9CPS. These strains were previously engineered via chromosomal integration of a Cas678-TniQ containing expression module. The four genes \u003cem\u003ecas678\u003c/em\u003e-\u003cem\u003etniQ\u003c/em\u003e were constructed into two expression cassettes \u003cem\u003etniQ\u003c/em\u003e-\u003cem\u003ecas7\u003c/em\u003e and \u003cem\u003ecas6\u003c/em\u003e\u0026ndash;\u003cem\u003ecas8\u003c/em\u003e, each regulated by different constitutive promoters and integrated into the \u003cem\u003elacA\u003c/em\u003e locus of \u003cem\u003eB. subtilis\u003c/em\u003e. Within the same expression cassette, the two gene units were linked in tandem by inserting an endogenous \u003cem\u003eB. subtilis\u003c/em\u003e RBS sequence (AAGGAGTGTCAAGA). In strains WS9CPG and WS9CPS, the expression cassette containing TniQ and Cas7 proteins are expressed under the control of promoters P\u003csub\u003e\u003cem\u003egsiB\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003esunA\u003c/em\u003e\u003c/sub\u003e, respectively, while the expression cassette containing Cas8 and Cas6 proteins is both driven by the promoter P\u003csub\u003e\u003cem\u003egroES\u003c/em\u003e\u003c/sub\u003e. We subsequently assembled the transposase expression plasmid pBSTnp and donor plasmid pBSDonor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).The transposase expression plasmid pBSTnp was generated by cloning genes encoding key transposition proteins (TnsA, TnsB, and TnsC) downstream of a tandem dual-promoter P\u003csub\u003e\u003cem\u003eHpaII\u003c/em\u003e\u003c/sub\u003e-P\u003csub\u003e\u003cem\u003eamyQ'\u003c/em\u003e\u003c/sub\u003e, followed by insertion into a modified pAD vector backbone containing the \u003cem\u003eB. subtilis\u003c/em\u003e replication origin and a chloramphenicol resistance marker. Meanwhile, we constructed the donor plasmid pBSDonor using the temperature-sensitive replication vector pE194. This plasmid contains two key functional units: a crRNA expression cassette driven by the P\u003csub\u003e\u003cem\u003eveg\u003c/em\u003e\u003c/sub\u003e promoter (designed to guide the CRISPR-Cas complex to specific genomic loci), and a spectinomycin resistance gene that serves as both the cargo and a selectable marker. The use of a temperature-sensitive replication origin in this donor plasmid facilitates its efficient clearance post-integration, thereby minimizing the likelihood of false-positive colonies during transposition validation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Development and optimization of a VchCAST-based programmable gene integration system in \u003cem\u003eBacillus subtilis\u003c/em\u003e. (A) Schematic diagrams of the engineered host strains WS9CPG and WS9CPS, along with the transposase and donor plasmids. The pBSTnp plasmid contains the P\u003csub\u003e\u003cem\u003eamyQ'\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003eHpaII\u003c/em\u003e\u003c/sub\u003e promoters, as well as the \u003cem\u003etnsA\u003c/em\u003e, \u003cem\u003etnsB\u003c/em\u003e, and \u003cem\u003etnsC\u003c/em\u003e genes. The pBSDonor plasmid includes a P\u003csub\u003e\u003cem\u003eveg\u003c/em\u003e\u003c/sub\u003e promoter, a crRNA composed of two repeat sequences (gray diamonds) and a spacer sequence (reddish-brown rectangle), and the RE-cargo (\u003cem\u003espe\u003c/em\u003e)-LE structure. (B) Illustration of the transposition experimental workflow of the VchCAST system in Bacillus subtilis. (C) Colony PCR validation. Detecting whether the system can achieve gene insertion in Bacillus subtilis strains WS9CPG and WS9CPS after resistance screening. (D) Sanger sequencing chromatograms of the upstream junction of the transposon in the RL integration orientation after performing transposition experiments in strain WS9CPS.\u003c/p\u003e \u003cp\u003eThe plasmids pBSTnp and pBSDonor were sequentially introduced into WS9CPG and WS9CPS host strains to establish two dual-plasmid editing systems. Following two rounds of subculturing at 37\u0026deg;C to mediate gene insertion, the bacterial cells were subjected to 51\u0026deg;C for 12 h for plasmid elimination (Figure. 1B). Single clones were obtained through resistance screening and verified by PCR amplification and sequencing, confirming precise integration of the cargo gene 49 bp downstream of the crRNA target site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Comparison of system efficiency demonstrated that when the P\u003csub\u003e\u003cem\u003esunA\u003c/em\u003e\u003c/sub\u003e promoter was used to transcript \u003cem\u003etniQ\u003c/em\u003e-\u003cem\u003ecas7\u003c/em\u003e genes, the resulting transposition efficiency was significantly higher than that of the P\u003csub\u003e\u003cem\u003egsiB\u003c/em\u003e\u003c/sub\u003e promoter-controlled system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Previous unpublished data from our laboratory using green fluorescent protein (GFP) to characterize promoter activity demonstrated that P\u003csub\u003e\u003cem\u003esunA\u003c/em\u003e\u003c/sub\u003e possesses stronger transcriptional activity than P\u003csub\u003e\u003cem\u003egsiB\u003c/em\u003e\u003c/sub\u003e, which we hypothesize to be the key factor enhancing the efficiency of the transposition system.\u003c/p\u003e\n\u003ch3\u003eOptimization of crRNA Promoter and TnsABC Expression\u003c/h3\u003e\n\u003cp\u003eAfter optimizing the expression strength of Cas protein and TniQ, we then attempted to improve transposition efficiency by optimizing expression of two core components: crRNA and transposase. Initially, we replaced the original crRNA promoter P\u003csub\u003e\u003cem\u003eveg\u003c/em\u003e\u003c/sub\u003e with six constitutive promoters\u0026mdash;P\u003csub\u003e\u003cem\u003esunA\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003ecitZ\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003egroESL\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003e43\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003eHpaII\u003c/em\u003e\u003c/sub\u003e, and P\u003csub\u003e\u003cem\u003eamyQ'\u003c/em\u003e\u003c/sub\u003e \u0026mdash;to regulate crRNA expression (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Following elimination of the pBSDonor plasmid, genomic DNA was extracted from colonies, and quantitative PCR (qPCR) was performed using primers specific to the genomic target site and the left (LE) and right (RE) ends of the transposon, with the \u003cem\u003eepr\u003c/em\u003e gene as an internal reference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Each sample was analyzed by qPCR through three parallel reactions targeting the internal reference gene, the RL integration direction, and the LR integration direction, respectively. The transposition efficiency for each orientation was calculated using the formula 2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e, where ΔCt represents the difference between the Ct value of the experimental reaction and that of the internal reference gene. The total transposition efficiency of the system was defined as the sum of the efficiencies for both orientations. Results demonstrated that the P\u003csub\u003e\u003cem\u003ecitZ\u003c/em\u003e\u003c/sub\u003e promoter achieved a transposition efficiency of 41% for the VchCAST system, indicating its superior capability in this context (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWe constructed a more stable transposition system by integrating the TnsABC transposase complex into the genome to circumventing plasmid instability. Transposition verification demonstrated that following plasmid clearance and antibiotic selection to reduce false-positive background, the single-plasmid VchCAST system achieved 80% transposition efficiency, significantly outperforming the dual-plasmid configuration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Optimization of the VchCAST System. (A) Schematic diagram of promoter optimization in the VchCAST system. The original promoter P\u003csub\u003e\u003cem\u003eveg\u003c/em\u003e\u003c/sub\u003e, responsible for regulating crRNA expression, was replaced with various alternative promoters. (B) Schematic illustration of the two possible integration orientations (RL and LR), along with the design of specific qPCR primer pairs used for their selective detection. (C) Evaluation of the influence of different promoters on transposition efficiency by qPCR. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 independent biological replicates. (D) Colony PCR verification. Detection of gene insertion efficiency of the VchCAST system after integration of the TnsABC expression cassette into the genome.\u003c/p\u003e \u003cp\u003eConsidering the large size of the TnsABC complex, we further tested a split-expression strategy by distributing TnsA, TnsB, and TnsC into two independent expression cassettes (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). TnsB, the largest and most critical subunit for DNA binding, was expressed under the P\u003csub\u003e\u003cem\u003egroES\u003c/em\u003e\u003c/sub\u003e promoter, while TnsA and TnsC were co-expressed from the original dual-promoter P\u003csub\u003e\u003cem\u003eHpaII\u003c/em\u003e\u003c/sub\u003e-P\u003csub\u003e\u003cem\u003eamyQ'\u003c/em\u003e\u003c/sub\u003e, with an endogenous \u003cem\u003eB. subtilis\u003c/em\u003e RBS sequence inserted between them. Contrary to expectations, this partitioned expression markedly reduced transposition efficiency (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), suggesting that the single expression cassette structure of the TnsABC complex may be more favorable for its proper expression.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual-site Transposition of VchCAST System in\u003c/b\u003e \u003cb\u003eB. subtilis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA notable advantage of the VchCAST transposon system lies in its capacity for simultaneous integration in multiple genome sites.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e This \"one-step\" integration strategy effectively circumvents the traditional sequential genetic manipulations, thereby substantially shortening the timeline for strain construction and optimization. Having established the system's high efficiency in single-site insertion, we further investigated its capability for simultaneously integration at dual distinct genomic loci.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Optimization of the VchCAST system for multi dual-site gene insertion. (A) Schematic of the donor plasmid designed for dual-site gene insertion. The original single-site crRNA has been replaced with a targeting RNA directed against the \u003cem\u003eepr\u003c/em\u003e and \u003cem\u003enprB\u003c/em\u003e loci. Additionally, the resistance gene is flanked by \u003cem\u003elox71\u003c/em\u003e and \u003cem\u003elox66\u003c/em\u003e sites, enabling subsequent Cre recombinase-mediated marker excision and thus supporting iterative rounds of integration. (B) The effect of overexpressing different host accessory factors on the transposition efficiency of the system was validated by colony PCR. The bar graph illustrates the single-site and dual-site insertion efficiencies for each experimental group. Insertion efficiency was calculated as the ratio of the number of positive clones to the total number of picked colonies in each experimental group. As the insertion efficiency was derived from a single round of colony PCR-based qualitative analysis, where only one set of colony picking and identification was performed per condition without experimental replicates, no error bars are shown in the graph.\u003c/p\u003e \u003cp\u003eWe selected two genomic sites (\u003cem\u003eepr\u003c/em\u003e and \u003cem\u003enprB\u003c/em\u003e) and constructed a dual-target donor plasmid. Furthermore, the spectinomycin resistance gene on the donor plasmid was flanked by \u003cem\u003elox71\u003c/em\u003e and \u003cem\u003elox66\u003c/em\u003e sites, which can be recognized by Cre recombinase. This design enables, upon successful gene integration, the removal of the \u003cem\u003espe\u003c/em\u003e resistance marker via the Cre-\u003cem\u003elox\u003c/em\u003e system, thereby allowing subsequent rounds of transformation with new donor plasmids for stacking additional insertion sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, during the initial assessment of dual-site transposition efficiency, while multiple clones with single-site insertions were obtained, no strains exhibiting simultaneous integration at both loci were identified. This outcome may indicate that under current conditions, the system's efficiency remains insufficient to support robust dual-site gene integration.\u003c/p\u003e \u003cp\u003eTo enhance the efficiency of dual-site simultaneous insertion, this study systematically screened host factors that may positively regulate the transposition process. Among these, the DNA-binding protein IhfA/B, as an integration host factor, can induce DNA conformational changes and influence recombination processes;\u003csup\u003e34\u003c/sup\u003e the HU protein subunits HupA and HupB have been reported to play a role in phage transposition;\u003csup\u003e35, 36\u003c/sup\u003e the transcriptional regulator BmrR, belonging to the MerR family, may indirectly modulate the expression of genes associated with transposition system function by inducing conformational changes in the DNA of target gene promoter regions;\u003csup\u003e37\u003c/sup\u003e the DNA-binding and -bending protein Fis is frequently involved in site-specific recombination and transcriptional regulation;\u003csup\u003e38\u003c/sup\u003e and the ATP-dependent protease ClpX may influence the dissociation of post-integration transposition complex.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Based on this background, we selected these six potentially relevant factors for investigation. By constructing their respective overexpression systems, followed by two rounds of passage culture and antibiotic screening, the transposition efficiency was systematically analyzed using colony PCR combined with nucleic acid gel electrophoresis. The results indicated that among the 24 picked monoclonal colonies, strains with single-site insertions were observed for all six factors, with varying numbers of insertions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Supplementary Figure S2). In the Fis overexpression group, only one colony exhibited dual-site gene insertion, while the HupB overexpression group yielded five colonies with dual-site insertions. However, no colonies with simultaneous dual-site insertion were obtained in the IhfA/B, ClpX, or HupA overexpression groups, possibly due to the relatively low transposition efficiency of the system when these factors are overexpressed. Notably, BmrR overexpression demonstrated the highest dual-targeting efficiency: six out of 25 monoclonal colonies successfully achieved simultaneous integration at both the \u003cem\u003eepr\u003c/em\u003e and \u003cem\u003enprB\u003c/em\u003e genomic sites, indicating its significant role in enhancing dual-site integration capability.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSite-Directed Mutagenesis of Transposase TnsB\u003c/h2\u003e \u003cp\u003eDuring natural evolution, the wild-type CRISPR-associated transposase (CAST) system, shaped by the survival needs of the host strain, does not exhibit the highest transposition efficiency. Excessively high transposition efficiency may impose an additional burden on the strain.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Therefore, the CAST system likely balances resource competition and survival pressure while maintaining essential functions and host viability, thereby achieving evolutionary stability. To overcome this limitation in the transposition capability of the wild-type system, we employed a semi-rational design strategy to engineer the CAST system with the aim of enhancing its transposition efficiency.\u003c/p\u003e \u003cp\u003eAs the key catalytic enzyme in the transposition process, TnsB is responsible for specifically recognizing transposon terminal sequences, cleaving target DNA strands, and mediating the connection between transposons and target sites. Its catalytic efficiency directly influence the overall integration capacity of the system. In this study, we implemented a computer-aided semi-rational protein design strategy to engineer TnsB. To systematically identify potential optimization sites, we integrated the PROSS algorithm\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and HotSpot Wizard tool\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e to perform multi-scale analysis and beneficial mutation anticipating of the TnsB protein. Based on computer anticipation and structure analysis, we selected 17 candidate sites for subsequent experimental validation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Engineering TnsB variants to obtain an efficient gene integration transposase system. (A) Effect of TnsB mutants on the gene insertion efficiency of the system. The efficiency of gene insertion by the transposon system was detected by qPCR, and the optimized enhancement efficiency was calculated by taking the ratio of the experimental group to the control group and then comparing it to the control group. (B) Multiple sequence alignment analysis of TnsB protein. (C) Effect of a site-directed mutation at amino acid position 178 of TnsB on the system's gene insertion efficiency. (D) Evaluation of system gene insertion efficiency after integrating the dominant mutants into the genome. (E) Structural prediction and simulation of the \u003cem\u003eVch\u003c/em\u003eTnsAB complex. Structure of the \u003cem\u003eVch\u003c/em\u003eTnsAB complex predicted by AlphaFold3 (center), further simulated in a conformation bound to DNA. Recently resolved crystal structure of the \u003cem\u003ePse\u003c/em\u003eTnsAB complex (left) is shown for comparison.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e In the figure, different monomers are represented by different colors: TnsB tetramers are shown in dark green, blue, purple, and light green representing TnsB.1 to TnsB.4; TnsA tetramers are shown in gray, yellow, pink, and cyan representing TnsA.1 to TnsA.4. (Right) A close-up shows the mutated V178 residue (red), whose predicted position is adjacent to transposon DNA (orange), suggesting it may participate in DNA interaction. Data in (A) (C) (D) are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 independent biological replicates.\u003c/p\u003e \u003cp\u003eUsing a dual-plasmid expression system consisting of the pBSTnp plasmid carrying TnsABC transposase and the pBSDonor plasmid carrying the target gene as the wild-type control platform, we constructed seventeen TnsB mutants (S28D, C31D, A70P, V178F, G194K, S215Y, L216A, V216L, V227L, E290P, L304H, L326G, D356T, K399N, E421P, E424P, M493K) and evaluated their transposition efficiency. The transposition efficiency of each mutant was systematically assessed through antibiotic resistance screening and real-time quantitative PCR analysis. The experimental results showed that among the 17 candidate sites, eight mutants (C31D, V178F, V227L, E290P, L304H, M493K, E424P, and L326G) exhibited improved transposition efficiency, with the V178F mutant demonstrating the most significant enhancement\u0026mdash;an increase of 232.6% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eSubsequently, we performed sequence alignment analysis on the 178 site of TnsB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Figure S3). Based on the alignment results, this site was mutated to leucine (L), isoleucine (I), and arginine (R), and the functional effects of these mutants were validated through transposition assays. qPCR results showed that when the 178 site was mutated to phenylalanine (F), the system maintained the highest transposition efficiency, while the V178L mutant also demonstrated a substantial improvement, with its relative transposition efficiency increasing by 178.07% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo further verify the enhanced gene insertion efficiency of the screened TnsB mutants in the transposition system, we integrated the TnsABC expression cassettes carrying the two advantageous mutants, V178F and V178L, into the genome. After subculturing and antibiotic screening, qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) indicated that the transposition efficiencies of the V178F and V178L systems were 95.25% and 90.24%, respectively. Additionally, we selected 24 single clones for colony PCR validation. Agarose gel electrophoresis results showed that the system mediated by the V178F mutant yielded 24 positive clones, while the V178L-mediated system yielded 22 positive clones, both exceeding the control group (Supplementary Figure S3).\u003c/p\u003e \u003cp\u003eFinally, we utilized AlphaFold3 to simulate the structure of the TnsAB protein and its bound DNA, and mapped the corresponding mutant residues of TnsB onto the predicted structure of the TnsAB tetramer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Structural analysis revealed that this residue is located near the DNA strand, indicating that the mutation at this site may enhance integration efficiency by modulating protein-DNA interactions.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe VchCAST transposase system originating from \u003cem\u003eV. cholerae\u003c/em\u003e represents an emerging genome editing technology capable of precise integration of large DNA fragments without relying on DNA double-strand breaks and homologous recombination repair. This study developed a VchCAST-based gene integration tool suitable for \u003cem\u003eB. subtilis\u003c/em\u003e. Initially, we successfully established the type I-F VchCAST system from \u003cem\u003eV. cholerae\u003c/em\u003e in \u003cem\u003eB. subtilis\u003c/em\u003e. The system efficiency demonstrated strong correlation with the expression levels of core components, particularly evidenced by the enhanced transposition efficiency observed when the \u003cem\u003etniQ\u003c/em\u003e-\u003cem\u003ecas7\u003c/em\u003e gene were driven by the strong promoter P\u003csub\u003e\u003cem\u003esunA\u003c/em\u003e\u003c/sub\u003e. This finding underscores the importance of maintaining high expression levels of key components when reconstructing complex protein machinery in heterologous hosts.\u003c/p\u003e \u003cp\u003eThrough optimization of the crRNA promoter to P\u003csub\u003e\u003cem\u003ecitZ\u003c/em\u003e\u003c/sub\u003e, the system efficiency was elevated to 41%, and genomic integration of the TnsABC transposase operon further enhanced efficiency to 80%, likely attributable to gene integration being more stable than plasmid expression. A key finding was that a split-expression strategy for TnsABC led to a marked decrease in efficiency, demonstrating that co-expression of the three core components within a single operon is more favorable for the expression of the transposase complex.\u003c/p\u003e \u003cp\u003eFurthermore, through overexpression of transposition accessory factors, we confirmed the potential of the VchCAST system for simultaneous dual-site integration in \u003cem\u003eB. subtilis\u003c/em\u003e. Previous research has demonstrated that BmrR can bind effector molecules and insert between the key promoter regions (-35 and \u0026minus;\u0026thinsp;10 boxes), inducing bending of double-stranded DNA. This structural modification enables proper recognition by RNA polymerase, thereby indirectly facilitating transcriptional activation.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e We therefore hypothesize that BmrR's potent DNA-distorting capability may be related to the gene integration process mediated by transposable elements, enhancing the accessibility of multiple target sites to the transposase complex and consequently alleviating efficiency bottlenecks during multiple integration events. This discovery not only provides new perspectives for understanding transposon-host interactions but also establishes novel pathways for enhancing CAST system performance through host environment engineering.\u003c/p\u003e \u003cp\u003eFinally, through computer-aided design, we successfully enhanced the transposition performance of TnsB and obtained two potentially optimized variants, V178F and V178L. Based on these findings, subsequent multi-site transposition validation experiments will employ V178F as the preferred engineered variant to evaluate its integration efficiency in industrial settings, thereby providing theoretical foundations and technical support for the broader application of this transposition system in synthetic biology.\u003c/p\u003e \u003cp\u003eThis study demonstrates that through systematic engineering, the VchCAST-based system enables efficient genome editing in Gram-positive bacteria \u003cem\u003eB. subtilis\u003c/em\u003e, significantly expanding the host range of this technology and highlighting the potential of VchCAST as a high-efficiency multi-copy integration tool. Currently, we are actively advancing the practical application of this system in the field of biosynthesis. Future work will focus on developing a VchCAST gene integration tool that operates without antibiotic selection, supports multi-site gene insertion, and is suitable for industrial-scale applications. The preliminary direction involves optimizing transposase activity through rational protein design and modulating its interaction network with host endogenous factors, thereby systematically enhancing transposition efficiency. The ultimate goal is to enable direct screening of positive clones from mixed microbial populations with minimal passaging, completely eliminating reliance on antibiotic-dependent selection methods.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflicts of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work received financial support from the National Natural Science Foundation of China (32430081, 32272264, 22478155), the Natural Science Foundation of Jiangsu Province (BE2023686), the National Key Research and Development Program of China (2024YFF1106300), the Fundamental Research Funds for the Central Universities (JUSRP122012), Shenzhen Institute of Synthetic Biology Scientific Research Program (DWKF20210004).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSC: Conceptualization, methodology, investigation, formal analysis, and writing the original draft. XZ: methodology, formal analysis, and writing review \u0026amp; editing. SG: methodology, formal analysis, and writing review \u0026amp; editing. XY: methodology. JS: formal analysis and writing the original draft. YC: Visualization. YL: Visualization. HL: Visualization. JW: methodology, writing review, and funding acquisition. LS: Methodology, supervision, and project administration. KZ: Conceptualization, methodology, formal analysis, supervision, and writing review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKov\u0026aacute;cs \u0026Aacute;T. Bacillus subtilis. 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Nat Commun. 2020;11(1):6284. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-020-20134-y\u003c/span\u003e\u003cspan address=\"10.1038/s41467-020-20134-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacillus subtilis, Gene insertion, CRISPR-associated transposase, TnsB","lastPublishedDoi":"10.21203/rs.3.rs-8770286/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8770286/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBacillus subtilis\u003c/em\u003e is a pivotal model organism in both industrial biotechnology and scientific research, where the efficiency of its genetic engineering is very important. However, achieving highly efficient gene insertion in this bacterium remains a significant technical challenge. To address this, we aimed to develop a novel gene insertion tool in \u003cem\u003eB. subtilis\u003c/em\u003e. Building upon the \u003cem\u003eVibrio cholerae\u003c/em\u003e-derived \u003cem\u003eVch\u003c/em\u003eCAST system, we systematically optimized and successfully established a high-performance VchCAST system. The core components of this system include the TniQ-Cas678 complex, a guide RNA for precise targeting, and the TnsABC transposase complex responsible for DNA integration. Under antibiotic selection, screening and employing a strong promoter to drive crRNA expression increased the single-locus transposition efficiency to 41%. Subsequent genomic integration of the transposase operational unit further enhanced the efficiency to 80%. Moreover, we demonstrated that overexpressing the auxiliary factor BmrR enables simultaneous integration at two distinct genomic loci. Through protein engineering of the key transposase TnsB, we obtained optimized variants V178F and V178L with significantly enhanced activity, which improved the overall transposition efficiency by 232.6% and 178.07%, respectively. We then conducted transposition validation with the optimized system, achieving a site-specific gene insertion efficiency of approximately 95.25%. In conclusion, this study not only provides a robust gene insertion platform for \u003cem\u003eB. subtilis\u003c/em\u003e microbial cell factory engineering, but also stands as a valuable reference for the construction of gene insertion tool in other microbial.\u003c/p\u003e","manuscriptTitle":"Development of an Efficient Gene Insertion Tool in Bacillus subtilis Based on CRISPR-associated transposase systems","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-10 16:07:48","doi":"10.21203/rs.3.rs-8770286/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e0a9ef49-9be7-4c2a-aee3-8150758823b4","owner":[],"postedDate":"February 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-27T04:54:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-10 16:07:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8770286","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8770286","identity":"rs-8770286","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}