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. 2025 Jul 2;10(4):1215–1223. doi: 10.1016/j.synbio.2025.06.012

Highly efficient genome editing in Bacillus subtilis via miniature DNA nucleases IscB

Jie Gao 1, Hengyi Wang 1, Jingtao Sun 1, Hongjie Tang 1, Yuhan Yang 1, Qi Li 1,⁎
PMCID: PMC12304945  PMID: 40735060

Abstract

Existing CRISPR-based genome editing techniques for Bacillus subtilis (B. subtilis) are limited due to the large size of the cas gene. IscB, a recently reported DNA nuclease, is one-third the size of Cas9, making it a potential tool for genome editing; however, its application in B. subtilis remains unexplored. In this study, two IscB and enhanced IscB (enIscB)-based genome editing systems, named pBsuIscB and pBsuenIscB were established in B. subtilis SCK6, and their deletion efficiencies ranging from 13.3 % to 100 %. Compared to the pBsuIscB system, the pBsuenIscB system showed higher deletion efficiency, inducing the deletion of a large genomic fragment with a single ωRNA. Additionally, the pBsuenIscB system could integrate both single-copy and multi-copy mCherry genes in the B. subtilis SCK6 genome. Lastly, the pBsuenIscB system could efficiently conduct a second round of genome editing in B. subtilis SCK6. This study indicates that IscB can be used for genome editing in B. subtilis, enabling the efficient construction of engineered B. subtilis strains for large-scale biomolecule production.

Keywords: IscB, Bacillus subtilis, Genome editing, Large genomic fragment deletion, Gene integration

1. Introduction

Bacillus subtilis (B. subtilis) is a Gram-positive bacterium with strong protein secretion ability and no endotoxins [1]. It is a “generally recognized as safe” bacterium and is widely utilized for the industrial production of various chemicals and recombinant proteins [2,3]. Genome editing tools have been used for both research and industrial applications of B. subtilis [4,5].

Genome editing involves the modification of genomic DNA (gDNA) at specific locations by adding, modifying, or deleting genetic sequences. The RNA-guided DNA nuclease could cleave the genomic DNA under the guidance of guide RNA (gRNA) to generate a double-strand break (DSB) and the genome could be repaired by providing homologous arms, thus enabling precise genome editing [6]. Both single-plasmid [[7], [8], [9]] and dual-plasmid [[9], [10], [11], [12], [13]] genome editing systems have been established for Cas9 and Cpf1 nucleases in B. subtilis; however, both systems exhibit certain limitations. For example, a single gRNA enables the modification of a single gene or a short DNA fragment. In contrast, deleting large genomic fragments required either the use of two gRNAs via the CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats, CRISPR; CRISPR associated nuclease, Cas9) system [12] or the pre-integration of the cpf1 gene into the genome via the CRISPR-Cpf1 system, thereby making the process laborious and time-consuming [14]. In addition, single-plasmid systems are inefficient due to the large size of the cas gene and the repair templates. Although the cas gene and gRNA are separated from the repair template in the dual-plasmid system, the multi-plasmids transformation is complex and requires more sophisticated antibiotics for plasmids stability. To address the above limitations, the development of miniaturized gene editor was considered as a novel and alternative strategy.

IscB is a recently identified miniature RNA-guided DNA nuclease encoded by the IS200/IS605 superfamily transposon. It is only one-third the size (496 amino acids) of Cas9 (1368 amino acids) and is hypothesized to be its ancestor [15]. Similar to Cas9, IscB contains HNH and RuvC domains and can form a ribonucleoprotein complex after binding with the omega RNA (ωRNA). This ribonucleoprotein complex cleaves DNA by recognizing the target adjacent motif (TAM) sequence (5′-CAGGAA-3′) and the target sequence (16 bp) [16]. Some researchers have engineered IscB and its corresponding ωRNA derived from the human intestinal macrogenome to obtain an enhanced IscB version named enIscB, which further improves editing activity [17]. Additionally, some researchers have developed modified IscBs with high activity for use in humans and mouse cells [18,19]. A novel IscB, mined from the macro-genome database, was modified into IscB.m16∗-BE, which has a broader TAM sequence range (from 5′-MRNRAA-3′ to 5′-NNNGNA-3′) [20].

Although IscB has great potential in genome editing applications, especially in eukaryotes, its application in prokaryotes (e.g., B. subtilis) remains unexplored. Therefore, this study aimed to explore whether IscB and enIscB could be utilized for genome editing in B. subtilis SCK6. For this, the toxicity performance and DNA cleavage-ability of IscB and enIscB in B. subtilis SCK6 were first verified. Thereafter, the IscB- and enIscB-based genome editing systems, named pBsuIscB and pBsuenIscB, respectively, were developed, and gene deletion abilities were analyzed. Subsequently, the ability of the pBsuenIscB system to delete large genomic fragments (using a single ωRNA), integrate single-copy or multi-copy mCherry genes, and conduct a second round of gene editing in B. subtilis SCK6 was examined. This is the first study to successfully establish an IscB-based genome editing tool in B. subtilis, thereby expanding the genome editing toolbox for B. subtilis and facilitating the construction of B. subtilis cell factories.

2. Materials and methods

2.1. Strains and culture conditions

The strains used in this study are listed in Table S1. Escherichia coli (E. coli) DH5α was used for cloning, plasmid maintenance, and plasmid construction, while B. subtilis SCK6 was used for genome editing. The two bacterial strains were cultivated in LB medium (0.5 % [w/v] yeast extract, 1 % [w/v] tryptone, and 1 % [w/v] NaCl) at 37 °C and 220 rpm. LB medium supplemented with 50 and 20 μg/mL kanamycin was used to screen plasmids in E. coli and B. subtilis SCK6, respectively. Competent B. subtilis SCK6 cells were prepared in YN medium (0.7 % [w/v] yeast extract, 1 % [w/v] tryptone, 0.5 % [w/v] NaCl, and 0.3 % [w/v] beef extract) supplemented with 1.5 % [w/v] xylose. The strains were stored in 20 % glycerol at −80 °C and recovered by initially streaking on LB plates and then inoculating isolated colonies in LB broth at 37 °C and 220 rpm for 12 h. Uracil (10 mg/L) was added as required.

2.2. Reagents and enzymes

The restriction enzymes used in this study were purchased from Thermo Fisher Scientific (Waltham, MA, USA). DNA polymerase 2 × Phanta Flash Master Mix, used for high-fidelity DNA amplification, was purchased from Vazyme Biotechnology Co., Ltd. (Nanjing, China). The 2 × Es Taq Master Mix (Dye) (Jiangsu Cowin Biotech Co., Ltd., Taizhou, China), used for E. coli colony polymerase chain reaction (PCR), while the 2 × Rapid Taq Master Mix, used for B. subtilis SCK6 colony PCR, was obtained from Vazyme Biotechnology Co., Ltd. The plasmid extraction and DNA purification kit was obtained from Tiangen Biotech Co., Ltd. (Beijing, China). The ClonExpress One Step Cloning Kit, used for the assembly of plasmids, was purchased from Vazyme427 Biotechnology Co., Ltd. (Nanjing, China).

2.3. Plasmid construction

All the plasmids and primers used in this study are listed in Tables S1 and S2. The IscB and enIscB gene sequences and their corresponding ωRNAs were derived from IscB-ωRNA and enIscB-ωRNA plasmids, respectively. The pIscB plasmid was prepared by inserting IscB and its xylose-induced PxylA promoter (amplified from pHT43-XCR6) into a linearized pcrF19-NM2 [11] plasmid vector and by replacing the pUC18 replicon with the p15A replicon (lower copy number). The cleavage plasmid, pBsuIscB-ωRNA-targetX (targetX represents the target gene), was constructed by inserting the ωRNA and its constitutive Pveg promoter into the pIscB plasmid. The pBsuIscB-targetX plasmid was obtained by inserting the repair template (∼1 kb upstream and downstream of the homologous arms in the B. subtilis SCK6 genome) into pBsuIscB-ωRNA-targetX. The enIscB-related plasmids were constructed similarly. The integration plasmids were prepared by inserting the integration gene and its promoter between the upstream and downstream homologous arms of the target gene.

2.4. Competent cell preparation and plasmid transformation

The B. subtilis SCK6 stored at −80 °C was streaked on LB plates for activation. Subsequently, the single colonies obtained from streaking were inoculated into fresh LB medium. The culture was incubated at 37 °C, 220 rpm for 12 h. Then the overnight bacterial culture was transferred to a fresh YN medium containing 1.5 % xylose (initial OD600: 1.0) and further incubated at 37 °C and 220 rpm for 2 h to form competent cells. The prepared plasmids were mixed with 500 μL of competent cells, and the mixture was incubated at 37 °C and 220 rpm for 90 min and then spread on LB plates.

2.5. Analysis of nuclease toxicity and genome cleavage-ability

For toxicity analysis, 5 μL of pIscB or penIscB plasmid was mixed with 500 μL of B. subtilis SCK6 competent cells and incubated at 37 °C and 220 rpm for 90 min. After transformation, the mixture was spread on LB plates containing 3 % xylose and kanamycin (experimental group) and LB plates with only kanamycin (control group) and incubated overnight at 37 °C. The colony-forming units (CFU) were counted to calculate the plasmid transformation efficiency (CFU/μg). A significantly lower transformation efficiency of the experimental group compared to that of the control group indicated the toxicity of IscB/enIscB toward B. subtilis SCK6, while equivalent transformation efficiencies of the experimental and control groups indicated the non-toxicity of the corresponding protein.

The genome cleavage-ability of IscB and enIscB was analyzed similarly using pBsuIscB-ωRNA-targetX and pBsuenIscB-ωRNA-targetX plasmids, respectively. A significantly lower transformation efficiency of the experimental group compared to the control group indicated that IscB/enIscB had B. subtilis SCK6 genome cleavage-ability, while equivalent transformation efficiencies of the experimental and control groups indicated that the corresponding protein was unable to cleave the B. subtilis SCK6 genome.

2.6. Gene deletion and insertion analyses

Briefly, 10 μL of pBsuIscB-targetX/pBsuenIscB-targetX-transformed B. subtilis SCK6 competent cells (500 μL) were inoculated on LB plates containing 3 % xylose and 20 μg/mL kanamycin and cultured overnight at 37 °C. Thereafter, 15 transformants were randomly selected for colony PCR. The deletion primers were complementary to the ∼50 bp sequences upstream and downstream of the homology arms. The insertion primers were located within the integration gene and ∼50 bp downstream of the homologous arm. The PCR products were sequenced to verify the positive colonies, and the wild-type strain was used as the control. For the integration of multi-copy mCherry genes, its copy number was verified by quantitative PCR (qPCR). As pyrE deficiency conferred a uracil auxotrophic phenotype to B. subtilis cells, the screening plates for strains with pyrE gene deletion required the addition of uracil (10 mg/L).

2.7. Fluorescent intensity measurement

To avoid interference from the expression of the mCherry gene on the plasmid, the plasmids in the integrated strains were eliminated, and the genotypes were verified by PCR. After plasmid curing, the mCherry-integrated strains were cultured overnight in LB medium, and then 1 % of the culture was inoculated into 50 mL LB broth. Samples were taken at 4 h, 6 h, 8 h, 10 h, 12 h, 24 h and 48 h to measure the OD600 and fluorescence values. The fluorescence of mCherry was measured at 587 nm excitation wavelength and 610 nm emission wavelength, and the fluorescence intensity (FI/OD600) was calculated. Meanwhile, the mCherry fluorescence of the bacterium was observed after centrifugation. The wild-type B. subtilis SCK6 was used as the blank control.

2.8. Plasmid curing

To eliminate the pBsuenIscB editing plasmid, the edited colonies containing the pBsuenIscB plasmid was inoculated in 5 mL of LB broth supplemented with 20 μg/mL kanamycin and incubated overnight at 37 °C and 220 rpm. Approximately 10 μL of the cells were transferred into antibiotic-free LB medium and incubated at 50 °C and 200 rpm for 8 h. The bacterial sample was continued three times at 50 °C with 200 rpm for 8 h, and then diluted and spread on antibiotic-free LB plates and incubated overnight at 37 °C. The bacterial colonies were randomly picked and screened by spotting on LB plates with or without 20 μg/mL kanamycin. The colonies that were sensitive to kanamycin were considered to be cured of pBsuenIscB. The same method was used to eliminate pBsuIscB.

2.9. qPCR analysis

The strain with a single copy of the mCherry gene integrated at the spo0A site was used as the standard strain. The gDNA of the standard strain and the strains with the mCherry gene integrated at the ribosomal RNA (rrn) operon were extracted and diluted to prepare template samples. The dnaN gene was selected as the single-copy reference gene of the B. subtilis SCK6 genome. Specific primer pairs (dnaN qup/dnaN qdn and mCherry qup/mCherry qdn; Table S2) were designed to generate products of approximately 180 bp. The PCR mixture (20 μL) contained 10 μL of 2 × Taq Pro Universal SYBR qPCR Master Mix, 0.4 μL of forward and reverse primers (10 μM), 10 ng genome DNA, and H2O. The reaction condition was as follows: 95 °C for 30 s; 40 cycles of 95 °C for 10 s and 60 °C for 10 s; and 65–95 °C in 0.5 °C/5 s increments. The gene copy-numbers for all the samples were calculated using the Pfaffl method. All experiments were performed with three independent biological replicates.

2.10. Statistical analysis

All the statistical data were calculated by using GraphPad Prism v10.1.2, and all experiments were performed at least three times (n = 3 independent experiments). Data are shown as mean ± standard error mean (SEM). Comparison between the two groups was conducted using one-way analysis of variance (ANOVA).

3. Results and discussion

3.1. IscB showed genome cleavage-ability in B. subtilis SCK6

Successful genome editing requires that IscB be nontoxic to B. subtilis SCK6. Therefore, the plasmids encoding IscB (pIscB) and enIscB (penIscB) were transformed into B. subtilis SCK6 and the transformants were spread on LB plates containing 3 % xylose and kanamycin and LB plates containing only kanamycin. The number of surviving colonies was counted to calculate the transformation efficiency (Fig. 1A). The results showed that B. subtilis SCK6 strains containing both pIscB and penIscB could grow on LB plates with or without xylose. Additionally, both strains showed comparable transformation efficiencies, and the transformation efficiency of penIscB was slightly higher than that of pIscB. These findings indicate that IscB and enIscB were non-toxic to B. subtilis SCK6 (Fig. 1B and Fig. S1).

Fig. 1.

Fig. 1

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IscB showed genome cleavage-ability in B. subtilis SCK6. (A) The toxicity results of pIscB/penIscB (above) and genome cleavage results of pBsuIscB-ωRNA/penIscB-ωRNA (below) in B. subtilis SCK6. +xylose represents LB plates with 3 % xylose, and -xylose represents LB plates without 3 % xylose. (B) The toxicity results of pIscB/penIscB in B. subtilis SCK6. (C) The efficiency of the genome cleavage by using pIscB. (D) The efficiency of the genome cleavage by using penIscB. Error bars represent means ± SEM (n = 3). Statistical significance was determined by using one-way ANOVA: ns, not significant; ∗p < 0.05; and ∗∗p < 0.01.

Following toxicity analysis, the genome cleavage abilities of IscB and enIscB in B. subtilis SCK6 were analyzed. The ωRNA was inserted into pIscB and penIscB plasmids to produce pBsuIscB-ωRNA (MolecularCloud number: MC_0101504) and pBsuenIscB-ωRNA (MolecularCloud number: MC_0101505) cleavage plasmids, respectively. The cleavage plasmids targeting the pyrE, spo0A, or bpr gene were transformed into B. subtilis SCK6. The transformants of the experimental groups (cleaved genome) were spread on LB plates containing 3 % xylose and kanamycin, and the transformants of the control groups (uncleaved genome) were spread on LB plates containing only kanamycin (Fig. 1A). Notably, the transformation efficiency of the experimental groups was nearly 0 CFU/μg DNA, while that of the control groups was 103 CFU/μg DNA (Fig. 1C, D and Fig. S2–3). These findings indicate that both IscB and enIscB can cleave the B. subtilis SCK6 genome.

These findings revealed that IscB and enIscB were nontoxic to B. subtilis SCK6 and could also cleave the genome with high efficiency. Thus, it was implied that further achieving genome editing in this host with IscB and enIscB by providing a repair template was promising [17]. In addition, the TAM sequences of IscB and enIscB (5′-CAGGAA-3′) are longer than the PAM sequences of Cas9 (5′-NGG-3′) and Cpf1 (5′-TTTN-3′). Due to the longer TAM sequences, IscB and enIscB can localize the target locus more accurately and reduce the nonspecific binding. Consequently, they demonstrate higher specificity in identifying target sequences and are expected to decrease the off-target rate. Additionally, compared to Cas9, the IscB- and enIscB-based editing systems have indeed demonstrated relatively lower off-target rates in relevant host organisms [17]. Thus, IscB and enIscB having favorable application prospects in genetic engineering research and related application fields.

3.2. Establishing the IscB-based genome editing system in B. subtilis SCK6

To validate the feasibility and the genome editing efficiency of IscB and enIscB in B. subtilis SCK6, the non-essential genes pyrE, spo0A, and bpr genes were selected for deletion testing [11,12]. The upstream and downstream sequences (∼1 kb) of the homologous arms of the deletion genes were added to the pBsuIscB-ωRNA and pBsuenIscB-ωRNA plasmids to obtain the pBsuIscB (MolecularCloud number: MC_0101506) and pBsuenIscB (MolecularCloud number: MC_0101507) genome editing systems, respectively. Then, the genome editing systems were transformed into B. subtilis SCK6 and 15 transformants were randomly selected to assess gene deletion (Fig. 2A). The TAM and N16 sequence data for pyrE, spo0A, and bpr genes are provided in Fig. 2B. The gene deletion efficiencies of the pBsuIscB system were 93.33 % (pyrE), 13.33 % (spo0A), and 100 % (bpr), while those of the pBsuenIscB system were 26.67 % (pyrE), 100 % (spo0A), and 100 % (bpr), respectively (Fig. 2C). The gene deletion in mutant strains was further verified by DNA sequencing (Fig. 2D–G).

Fig. 2.

Fig. 2

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The IscB-based genome editing system was established in B. subtilis SCK6. (A) Schematic representation of gene deletion in B. subtilis SCK6 based on the pBsuIscB/pBsuenIscB system. (B) The target sequences of different target genes in B. subtilis SCK6. (C) Gene deletion efficiency of the pBsuIscB/pBsuenIscB system. (D) DNA gel images for PCR verification of spo0A, pyrE, and bpr gene deletion by the pBsuIscB system. (E) DNA sequencing results for the verification of spo0A, pyrE, and bpr gene deletion by the pBsuIscB system. Lengths of the deleted genes: Δspo0A, 804 bp; ΔpyrE, 651 bp; and Δbpr, 4,302 bp. (F) DNA gel images for PCR verification of spo0A, pyrE, and bpr gene deletion by the pBsuenIscB system. (G) DNA sequencing results for the verification of spo0A, pyrE, and bpr gene deletion by the pBsuenIscB system. Lengths of the deleted genes: Δspo0A, 804 bp; ΔpyrE, 651 bp; and Δbpr, 4,302 bp.

These findings indicate that the pBsuIscB and pBsuenIscB systems can achieve gene deletion in B. subtilis SCK6 with 13.3 %–100 % efficiency. Further analysis revealed that the pBsuenIscB system had higher editing efficiency compared with the pBsuIscB system. It's speculated that this might be related to the fact that enIscB was engineered from IscB, since the editing activity of enIscB was indeed higher than that of IscB in mammals [16]. We compared the reported gene editing methods in B. subtilis in recent years, mainly targeting Cas9 and Cpf1 based methods (Table S3). The results demonstrated that the CRISPR-Cas9, CRISPR-Cpf1, and pBsuenIscB systems (whether delivered via single-plasmid, dual-plasmid, or genome-integrated approaches) are all capable of efficiently deleting single gene or short sequence. And pBsuenIscB system was equivalent to that of the CRISPR system [21]. As the pBsuenIscB system exhibited higher editing efficiency, it was employed for further analyses.

3.3. pBsuenIscB system achieved large genomic fragment deletion in B. subtilis SCK6

Both pBsuIscB and pBsuenIscB systems exhibited the ability to delete regular genes (pyrE, spo0A, and bpr) in the B. subtilis SCK6 genome. However, in practice, the improvement of microbial cell factory efficiency involves the deletion of non-essential regions to reduce transcription costs and eliminate competing pathways, and these non-essential regions are tens of kilobases or even more than 100 kb in size [[22], [23], [24]]. Currently,the methods for deleting large genomic fragments based on CRISPR-Cas9 [12] and CRISPR-Cpf1 [14] in B subtilis are time-consuming and laborious.

To evaluate the potential of the pBsuenIscB system for deleting large genomic fragments in B. subtilis SCK6, the pps operon (37.7 kb), which is non-essential for cell growth, was chosen as the target. The homology arm (∼1 kb upstream and downstream) and the single ωRNA (targeting the middle of the pps operon) were added to penIscB to yield the pBsuenIscB-pps editing plasmid (Fig. 3A). The editing plasmid was transformed into B. subtilis SCK6 and 15 transformants were randomly selected for PCR verification. Since the primers were designed for the sequence outside the homology arm on the genome, a 2 kb band remained, following because of the deletion of the pps operon. In contrast, the band of the wild-type strain was approximately 40 kb and no results appeared. These results suggest that the pBsuenIscB system successfully deleted the pps operon with 100 % efficiency (Fig. 3B). To further verify whether the pBsuenIscB system can delete larger genomic fragments, a 169.9 kb genomic fragment was selected for deletion (Fig. 3C). Notably, the pBsuenIscB system successfully deleted this 169.9 kb fragment with 40 % efficiency (Fig. 3D).

Fig. 3.

Fig. 3

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The pBsuenIscB system enabled the deletion of large genomic fragments in B. subtilis SCK6. (A) Schematic diagram of the target site and homologous arms of the 37.7 kb pps operon. (B) DNA gel image and sequencing results verifying the deletion of the pps operon by the pBsuenIscB system. (C) Schematic diagram of the target site and homologous arms of the 169.9 kb genomic fragment. (D) DNA gel image and sequencing results verifying the deletion of the 169.9 kb genomic fragment.

These findings indicate that the pBsuenIscB system can delete genomic DNA fragments of 37.7 kb and 169.9 kb, using only a single ωRNA. However, the Cas9-based two-plasmid system required sequential plasmids transformation and design of two gRNAs, resulting in a more complicated editing process [12]. While the Cpf1 system could utilize a single crRNA for large DNA fragment deletion, but it required the integration of Cpf1 encoding gene into the genome in advance [14]. Thus the Cpf1-based method was complex and time-consuming, and its efficiency was lower than pBsuenIscB. Overall, the use of a single ωRNA in the pBsuenIscB system reduced complexity, simplified plasmid construction, and improved operational efficiency.

3.4. Gene integration can be achieved in B. subtilis SCK6 by pBsuenIscB system

Currently, plasmids are mainly used in industrial production to increase the synthesis of target products. However, plasmids rely on antibiotics to prevent their destabilization in host bacteria. To achieve stable gene expression, the gene expression cassette should be integrated into the host genome [25]. This study investigated the gene integration efficiency of the pBsuenIscB system at different loci and the gene expression efficiency at different loci. The mCherry gene, expressing the red fluorescent mCherry protein, was used as the cargo gene for integration. First, genes have been targeted for deletion or integration in previous studies were selected as candidates. Second, to investigate the expression efficiency of mCherry at different genomic loci in B. subtilis, sites evenly distributed across the B. subtilis genome were further chosen [11,26]. Therefore, these six genes spo0A, bpr, ctc, epr, nprB, and thrC were finally selected as integration sites for the mCherry gene (Fig. 4A).

Fig. 4.

Fig. 4

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Integration of the mCherry gene at different loci in the B. subtilis SCK6 genome. (A) Schematic representation of mCherry gene integration at ctc, epr, spo0A, bpr, thrC, and nprB loci and the corresponding sequencing results. (B) Confirming the elimination of plasmids in the integrated strains. (C) The genotypes of the integrated strains following plasmid elimination. (D) Fluorescence intensity and OD600 values of the integrated strains. ck: wild-type SCK6 strain. (E) Fluorescence photos of the integrated strains.

The mCherry gene (controlled by the Pspl promoter) was inserted between the upstream and downstream homologous arms of pBsuenIscB-targetX to obtain the pBsuenIscB-targetX-mCherry integrating plasmid, which was then transformed into B. subtilis SCK6. The pBsuenIscB system integrated mCherry at ctc, epr, spo0A, bpr, thrC, and nprB sites with 6.6 %, 6.6 %, 20 %, 6.6 %, 26.6 %, and 13.3 % efficiency, respectively (Fig. S4). Gene integration was verified by sequencing (Fig. 4A). As for the efficiencies of knock-in are among 6.6 %–26.6 %, it was speculated that the reason is as follows: gene integration requires the replacement of the target sequence, however gene deletion only requires the removement of the target sequence, resulting in more complex process and decreasing the efficiency. Moreover, other study also showed that the integration efficiency based on the single-plasmid editing system is lower than the deletion efficiency, at only 9 % [14]. While the pBsuenIscB system could achieve the integration at multiple loci with higher efficiency.

The effect of different integration sites on mCherry gene expression was explored in B. subtilis SCK6. The plasmids were eliminated from all six mCherry integration strains (Fig. 4B), and the genotypes were verified by PCR (Fig. 4C). The OD600 values and fluorescence intensities of each integrated strain at different culture times indicated that the fluorescence intensity gradually increased with an increase in culture duration. The fluorescence intensity reached the highest level at 48 h of culture, followed by 24 h of culture. Among the six integrated strains, the spo0A site-integrated strain showed a decrease in growth compared to the wild-type SCK6 strain (Fig. 4D). Besides, all integrated strains were photographed for fluorescence at the corresponding time points. The spo0A site-integrated strain showed nearly no red fluorescence compared to the other five integrated strains, which showed a gradual increase in red fluorescence with an increase in culture duration (Fig. 4E). These findings indicate that mCherry expression increased within 48 h after integration at the ctc, epr, bpr, thrC, and nprB sites in the integrated strains, which is consistent with the measured fluorescence values and the OD600 readings.

A previous study found that the B. subtilis genome has 10 copies of the rrn operon (encoding bacterial ribosomal 23S, 16S, and 5S rRNAs), which is essential for its growth. However, B. subtilis can maintain its growth rate at an rrn copy number >2 [27]. Therefore, it is feasible to integrate a cargo gene at the rrn operon without affecting B. subtilis growth. To increase the expression of the mCherry gene, multiple copies of the mCherry gene were integrated into the rrn operon (Fig. S5A). The pBsuenIscB-rrn-mCherry integrating plasmid was transformed into B. subtilis SCK6 and 69 transformants were randomly screened for positive colonies. The pBsuenIscB system successfully integrated the mCherry gene into the rrn operon with 39.7 % efficiency (Fig. S5B–C). The multi-copy mCherry gene strain was named Mut-MC-mCherry, and the strain with a single-copy mCherry gene at the spo0A site was named Mut–SC–mCherry. The gDNA was extracted from eight Mut-MC-mCherry strains (with multiple copies of the mCherry gene) and the Mut–SC–mCherry strain (with a single copy of mCherry gene at the spo0A site) for qPCR; the latter was used as the standard, and the dnaN gene was used as the reference gene. Notably, strains 24 and 47 had integrated mCherry copy numbers of 8 and 3, respectively, while the other six strains had a single copy of the mCherry gene (Fig. S5D). Attempts to eliminate the plasmids within the multi-copy integrated strains were unsuccessful, thus, fluorescence intensity testing was not performed at this stage.

There are two main methods for multi-copy gene integration in B. subtilis. The first method involves the sequential integration of single genes [28], which is time-consuming, laborious, and has low efficiency. The second method involves the integration of the crtMN operon (encoding xanthophyll) at three sites in the B. subtilis genome and the simultaneous integration of up to three copies of the target gene at these sites using the CRISPR-Cas9 system [29]. In this study, all 10 copies of the rrn operon were simultaneously targeted with only one ωRNA to randomly achieve the integration of 3 and 8 copies of the mCherry genes in the B. subtilis SCK6 genome. This method can be used to enhance the integration rate of multi-copy genes.

This study demonstrated that the pBsuenIscB system can integrate single-copy genes as well as multiple-copy genes simultaneously at the rrn site and can achieve stable cargo gene expression.

3.5. pBsuenIscB system achieved plasmid curing and second round of genome editing in B. subtilis SCK6

Plasmid curing is a prerequisite for phenotype testing and the second round of genome editing [30]. The pBsuenIscB-bpr plasmid was cured from was selected as the target. The correctly edited B. subtilis SCK6Δbpr strain was inoculated into antibiotic-free LB medium and then incubated at 50 °C with shaking at 200 rpm for 8 h. This process of incubation and subculturing was repeated for 3 times. Subsequently, the bacterial solution was taken and diluted for spreading to obtain single colonies, of which 52 were subjected to a kanamycin spotting test (Fig. 5A). Of the 52 colonies, 48 colonies were sensitive to kanamycin, suggesting that pBsuenIscB-bpr had been eliminated with 92.3 % efficiency (Fig. 5B).

Fig. 5.

Fig. 5

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The pBsuenIscB system was cured and the second round of genome editing was accomplished successfully. (A) Operational procedures for the elimination of the pBsuenIscB plasmid. (B) Kanamycin spotting test to verify pBsuenIscB-bpr plasmid curing in the B. subtilis SCK6Δbpr strain. (C) DNA gel image after the second round of genome editing at the spo0A site using the pBsuenIscB system; strains 5 and 11 showed successful gene editing. (D) DNA gel image verifying the deletion of the bpr and spo0A genes in strains 5 and 11.

For the second round of genome editing, the pBsuenIscB-spo0A editing plasmid was transformed into the above B. subtilis SCK6Δbpr strain with the plasmid already cured. The pBsuenIscB system successfully achieved the second round of genome editing at the spo0A site with 13.3 % efficiency (Fig. 5C). The deletion of bpr and spo0A genes was further verified by PCR (Fig. 5D). We repeated the second round of gene deletion (including spo0A and pyrE) in the SCK6Δbpr strain, the editing efficiency of the spo0A gene was 33.3 % (Fig. S6A), which was higher than the previous editing efficiency of 13.3 %. While the results of the second round editing of pyrE gene showed consistent with the previous editing efficiency (Fig. S6B). Statistical analysis showed no significant difference in editing efficiencies between the first and second rounds of gene editing.

This research successfully cured the editing plasmid pBsuenIscB-bpr and achieved the second round of genome editing using the pBsuenIscB system. It verified the feasibility of the system for multi-target genomic editing and provided a basis for modifying engineered strains with the pBsuenIscB system.

CRediT authorship contribution statement

Jie Gao: Writing – original draft, Methodology, Validation, Investigation, Data curation. Hengyi Wang: Writing – review & editing, Formal analysis. Jingtao Sun: Formal analysis, Writing – review & editing. Hongjie Tang: Writing – review & editing, Methodology, Formal analysis. Yuhan Yang: Investigation, Data curation. Qi Li: Formal analysis, Writing – review & editing, Methodology, Supervision.

Declaration of competing 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.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Number: 32402894) and the Sichuan Science and Technology Program (Number: 2024NSFSC0373). We would like to thank Prof. Hui Yang and Yingsi Zhou for providing the pIscB/penIscB-ωRNA related plasmids and Prof. Long Liu for providing the pHT-XCR6 and pcrF19-NM2 plasmids.

Footnotes

Peer review under the responsibility of Editorial Board of Synthetic and Systems Biotechnology.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.synbio.2025.06.012.

Appendix A. Supplementary data

The following is the supplementary data to this article:

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