Methods

Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase

Kaifeng Li, Dongbo Cai, Zhangqian Wang, Zhili He, Shouwen Chen
Maia Kivisaar, Editor
DOI: 10.1128/AEM.02608-17

ABSTRACT

Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN, which codes for nattokinase in Bacillus subtilis, was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-SsacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC. Finally, the engineered strain DWc9nΔ7 (Δepr ΔwprA Δmpr ΔaprE Δvpr ΔbprA ΔbacABC), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis. This tool could be applied to strain improvement for future research.

IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

INTRODUCTION

Serving as a kind of natural immune system in many bacteria and archaea, the clustered regularly interspaced short palindromic repeat (CRISPR) system is used to prevent the invasion of foreign DNA (1–3). In recent years, the type II-A CRISPR-Cas9 system has been widely used in genome editing due to its high efficiency, ease of design, short manufacturing time, and low cost (4, 5). The type II-A CRISPR-Cas9 system consists of two main elements, a Cas9 DNA endonuclease and a targeted chimeric single-guide RNA (sgRNA) (see Fig. S1 in the supplemental material) (6–8). The Cas9 endonuclease creates double-strand breaks at the target site under the guidance of a specific sgRNA (9). The cleavage site then is repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) (10–12). Until now, Cas9 endonuclease coupled with NHEJ has been widely applied to genome editing in mammals, eukaryotes, and some bacteria (13, 14).

Current genome editing applications based on the type II-A CRISPR-Cas9 system in bacteria are classified into two categories: Cas9-mediated genome editing and Cas9 nickase (Cas9n)-mediated genome editing (15). The CRISPR-Cas9 system has been applied to genome editing, epigenome studies (16), and disease treatments (17). Moreover, the plasmid-based CRISPR-Cas9 system and NHEJ have been used for double-gene disruption (18), inactivation of bacterial genes, and large DNA fragment deletion (19). However, there are some limitations to the CRISPR-Cas9 system. Specifically, this system is not suitable for all bacteria, as the lack of or low expression of major NHEJ components, including Ku protein (20, 21), ATP-dependent DNA ligase (22, 23), and DNA polymerase LigD (24, 25), can result in death of the bacteria after cleavage. Cas9n is more useful in these strains, since the lethality effect can be avoided (4).

Cas9n D10A is a mutant of Streptococcus pyogenes Cas9 that causes single-strand DNA breaks. When combined with the repair of cleavage sites by the donor template, this enzyme can be useful for genome editing. Cas9n combined with HDR is considered a more precise genome editing tool, since only the fragment between the donor templates can be modified. Furthermore, to decrease off-target effects associated with CRISPR-Cas9, a new double-nicking strategy involving a pair of sgRNAs was designed using Cas9n. Using this system, the off-target mutation frequency was decreased by 50- to 1,500-fold in human cells (26). Therefore, CRISPR-Cas9n has been used to avoid bacterial death in NHEJ-deficient strains, precisely edit the genome outside the target sequence, and decrease the frequency of off-target mutations. Cas9n-mediated genome editing has been shown to be an efficient and precise tool in Clostridium cellulolyticum (4), Escherichia coli (27), and Lactobacillus casei (28), but the genome editing strategies related to Cas9n are rarely reported in Bacillus.

Bacillus licheniformis is an industrial bacterial strain with high genetic diversity that has been applied to the production of various biochemicals. B. licheniformis has also been shown to be an efficient host for heterologous protein expression due to its excellent secretion capabilities and because it is nontoxic (29). However, a lack of efficient genome editing tools has greatly hindered its strain development. In this study, B. licheniformis DW2 was used to develop a fast, efficient, and systematic genome editing method based on the CRISPR-Cas9n system. Single-gene knockout, large DNA fragment deletion, simultaneous disruption of two genes, and single-gene integration strategies were developed using this system. Moreover, we were able to significantly increase nattokinase activity in the recombinant strain DWc9nΔ7 constructed using this system. This study describes an efficient and systematic genome editing system based on CRISPR-Cas9n that is a promising tool for strain improvement in the future.

RESULTS

Construction of the recombinant strain integrating Cas9n.In this study, the Cas9 nickase gene mutated from S. pyogenes cas9 was used to construct the CRISPR-Cas9n system in B. licheniformis DW2. To confirm the expression of Cas9n in B. licheniformis, an expression plasmid containing the gene encoding Cas9 nickase was constructed using the pHY300PLK vector and named pCas9n. B. licheniformis DW2 then was transformed with pCas9n, and the resulting recombinant strain was named DW2/pCas9n. B. licheniformis DW2 was also transformed with pHY300PLK to construct the control strain DW2/pHY300. These two strains were cultivated in LB medium, and their intracellular proteins were identified with SDS-PAGE. A 140-kDa protein band is present in the DW2/pCas9n strain but not in the DW2/pHY300PLK control strain (Fig. 1A). The size of this band was the same as that of Cas9n, which indicates that Cas9n was successfully expressed in B. licheniformis DW2.

FIG 1
FIG 1

Expression and integration of Cas9 nickase in B. licheniformis. (A) SDS-PAGE analysis of the intracellular proteins in DW2/pHY300 and DW2/pcas9n. Lane M, 200-kDa protein marker (200, 150, 120, 100, 85, 70, 60, and 50 kDa); lane 2, DW2/pHY300; lane 3, DW2/pcas9n. The arrow indicates the Cas9 nickase. (B) Confirmation of Cas9n integration strain by PCR amplification. Lane M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 1, PCR product of DW2 using Cas9n-F and Cas9n-R (no DNA bond with this control strain); lane 2, PCR product of DWc9n using Cas9n-F and Cas9n-R (4,933 bp). (C) The construction procedure for the Cas9n integration expression strain DWc9n.

To decrease the capacity of the plasmid and make electroporation easier, the Cas9n expression cassette driven by the P43 promoter was integrated into the genome of B. licheniformis DW2 (Fig. 1B). Since the Cas9n expression cassette is too large for gene integration, we integrated the expression cluster into the chromosome of B. licheniformis DW2 using a two-step protocol (Fig. 1C), and the final strain was named DWc9n.

Single-gene knockout using the CRISPR-Cas9n system.In our previous research, the yvmC gene was confirmed to be essential for the synthesis of pulcherrimin, a red pigment produced by B. licheniformis DW2 (30). In the current study, the yvmC gene was chosen as a target gene to measure the effectiveness of single-gene editing by CRISPR-Cas9n. Our results showed that yvmC could be successfully deleted with an efficiency of 100% using upstream and downstream homology arms of 1.0 kb (Fig. 2A and B). To determine the optimal size of homology arms for high editing efficiency, sgRNA expression cassettes containing homology arms of 0.1, 0.2, 0.3, 0.5, and 0.7 kb were amplified for single-gene deletion. The editing efficiency increased with increasing homology arm size (Fig. 2C). However, the yvmC gene was deleted with high efficiency (51.4%) even with the shortest homology arms (0.1 kb).

FIG 2
FIG 2

Precise deletion of single gene and efficiency evaluation. (A) The strategy for knocking out yvmC in B. licheniformis by CRISPR-Cas9n system. (B) Confirmation of the yvmC-deficient strain by PCR amplification. Lane M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 1, PCR product of DWc9n using Y-F/Y-R (3,122 bp); lane 2, PCR product of DWc9n using M-F/Y-R (1,792 bp); lane 3, PCR product of DWc9n△yvmC using Y-F/Y-R (2,427 bp); lane 4, PCR product of DWc9n△yvmC using M-F/Y-R (no DNA bond for lacking the target sequence of the primer M-F). (C) Effects of different sizes of homologous arms on the efficiency of yvmC deletion.

Simultaneous deletion of two genes using the CRISPR-Cas9n system.Although the CRISPR-Cas9n system has previously been applied to single-gene modifications (4, 28, 31), few studies have reported multiple-gene deletion using the CRISPR-Cas9n system in Bacillus. Here, we attempted to simultaneously delete two genes using the novel plasmid pHY300-ΔeprΔwprA (Fig. 3A). sgRNAs were targeted to the genes epr and wprA, leading to cleavage by Cas9n at both sites on the genome. Donor templates then were used to simultaneously disrupt these two genes. Based on our results, the deletion efficiency of epr and wprA was 75.4% and 11.6%, respectively (Fig. 3B). The efficiency of the double-gene disruption was 11.6% (Table 1).

FIG 3
FIG 3

Precise disruption of epr and wprA simultaneously. (A) Plasmid construction procedure for double gene (epr and wprA) deletion. (B) Confirmation of epr- and wprA-deficient strain by PCR amplification using primers △epr-Y-F/Y-R and △wprA-Y-F/Y-R. Lane M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 2, PCR product of DWc9n using primers △epr-Y-F/Y-R (2,328 bp); lane 3, PCR product of DWc9n△epr△wprA using primers △epr-Y-F/Y-R (1,428 bp); lane 4, PCR product of DWc9n using primers △wprA-Y-F/Y-R (3,802 bp); lane 5, PCR product of DWc9n△epr△wprA using primers △wprA-Y-F/Y-R (2,805 bp).

View this table:
TABLE 1

Possible efficiency of various CRISPR-Cas9n plasmids

Large DNA fragment deletion using the CRISPR-Cas9n system.A novel strategy was designed to knock out a large DNA fragment in B. licheniformis. Bacitracin is a broad-spectrum polypeptide antibiotic produced by B. licheniformis DW2 that is synthesized by nonribosomal peptide synthetases (32). We chose the bacitracin synthase gene cluster bacABC, which is ∼42.7 kb, as the target gene for large DNA fragment editing using the CRISPR-Cas9n system. The plasmid pHY300-ΔbacABC, containing two specific sgRNAs targeting both ends of the same strand of the bacABC gene and linked to the donor template, was constructed. This allowed for the target sites to be cut by the Cas9n enzyme and the large bacABC fragment to be deleted via the HDR pathway (Fig. 4A). The deficient strain was verified using specific primers targeted to both ends of the homology arms. Colony PCR and DNA sequencing confirmed that the bacABC gene cluster was successfully deleted (Fig. 4B and C). The gene editing efficiency was 79.0% (Table 1). Thus, our results confirm that the CRISPR-Cas9n system can be applied to large DNA fragment editing.

FIG 4
FIG 4

Precise deletion of a large DNA fragment. (A) The construction procedure for the bacitracin synthase gene cluster bacABC-deficient strain. (B) Confirmation of bacABC-deficient strain by PCR amplification using primers △bacABC-Y-F/Y-R. Lane M, DL 5000 marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 1, PCR product of bacABC in DWc9n (too large to amplify); lane 2, PCR product of DWc9n△bacABC (1,977 bp). The verified primers are designed at both ends of bacABC (Y-F and Y-R) and the PCR product size is 1,977 bp in the bacABC-deficient strain, whereas no PCR product was found for the wild-type strain on gel electrophoresis. (C) Confirmation of bacABC-deficient strain by DNA sequence.

Gene integration into B. licheniformis using the CRISPR-Cas9n system.Nattokinase is widely used for the prevention and treatment of thrombus-related diseases. Here, the aprN expression cassette was integrated into the chromosome of DWc9n using the CRISPR-Cas9n system. The aprN expression cassette was inserted between upstream and downstream homology arms to be integrated into the chromosomal target site via the HDR pathway (Fig. 5A). Agarose gel electrophoresis (Fig. 5B) and DNA sequencing (Fig. 5C) results confirmed that the aprN expression cassette was successfully integrated into the chromosome of DWc9n with an efficiency of 76.5% (Table 1).

FIG 5
FIG 5

Precise insertion of aprN expression cassette. (A) The construction procedure for aprN integration strain. (B) Confirmation of aprN integration by PCR amplification using primers NK-Y-F/Y-R. Lane M, DL 5000 marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 2, PCR product of DWc9n using primers NK-Y-F/Y-R (1,592 bp); lane 3, PCR product of aprN integration strain using primers NK-Y-F/Y-R (2,927 bp). The star represents the aprN integration strain. (C) Sequencing analysis of integrated site in DWc9n.

Construction of a heterologous protein-expressing strain using the CRISPR-Cas9n system.B. licheniformis has been shown to be an efficient strain for heterologous protein expression due to its excellent secretion capability and because it is nontoxic. However, excessive protease might decrease the production of target protein. Here, six extracellular protease genes (epr, wprA, mpr, aprE, vpr, and bprA) and the bacitracin synthase gene cluster bacABC were deleted using the CRISPR-Cas9n system to construct the strain DWc9nΔ7. Colony PCR and DNA sequencing confirmed that these genes were successfully deleted (Fig. 6).

FIG 6
FIG 6

Confirmation of the extracellular protease gene-deficient strain by PCR amplification. Lane M, DL 5000 DNA marker (5,000, 3,000, 2,000, 1,500, 1,000, 750, 500, 250, and 100 bp); lane 2, PCR product of DWc9n using primers △epr-Y-F/Y-R (2,328 bp); lane 3, PCR product of DWc9n△7 using primers △epr-Y-F/Y-R (1,428 bp); lane 4, PCR product of DWc9n using primers △wprA-Y-F/Y-R (3,802 bp); lane 5, PCR product of DWc9n△7 using primers △wprA-Y-F/Y-R (2,805 bp); lane 6, PCR product of DWc9n using primers △mpr-Y-F/Y-R (2,054 bp); lane 7, PCR product of DWc9n△7 using primers △mpr-Y-F/Y-R (1,112 bp); lane 8, PCR product of DWc9n using primers △aprE-Y-F/Y-R (1,791 bp); lane 9, PCR product of DWc9n△7 using primers △aprE-Y-F/Y-R (945 bp); lane 10, PCR product of DWc9n using primers △vpr-Y-F/Y-R (1,882 bp); lane 11, PCR product of DWc9n△7 using primers △vpr-Y-F/Y-R (886 bp); lane 12, PCR product of DWc9n using primers △bprA-Y-F/Y-R (2,258 bp); lane 13, PCR product of DWc9n△7 using primers △bprA-Y-F/Y-R (742 bp); lane 14, PCR product of DWc9n using primers △bacABC-Y-F/Y-R (too large to amplify); lane 15, PCR product of DWc9n△7 using primers △bacABC-Y-F/Y-R (1,977 bp).

Nattokinase production in the engineered strain.The nattokinase expression plasmid pP43SNT-SsacC was obtained in our previous research (33). In the current study, DWc9nΔ7 and DW2c9n were transformed with pP43SNT-SsacC, resulting in the strains DWc9nΔ7/pP43SNT-SsacC and DW2c9n/pP43SNT-SsacC, respectively. DWc9nΔ7/pP43SNT-SsacC and DW2/pP43SNT-SsacC were cultured in nattokinase production medium. The growth of these two strains was not significantly different before 30 h (Fig. 7). After 30 h, the growth rate of DWc9nΔ7 was faster than that of DWc9n. Additionally, the biomass of DWc9nΔ7/pP43SNT-SsacC was higher than that of DWc9n/pP43SNT-SsacC. During the fermentation process, the maximum activity of DWc9nΔ7/pP43SNT-SsacC was 59.7 fibrinolytic units (FU)/ml at 36 h, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC at 42 h (47.5 FU/ml).

FIG 7
FIG 7

Time profiles of nattokinase production and cell growth in DWc9n/pP43SNT-SsacC and DWc9n△7/pP43SNT-SsacC strains.

DISCUSSION

In recent years, the CRISPR-Cas9 system has received widespread attention in the field of genome editing, owing to the fact that it is easy to operate and has high efficiency and a short manufacturing time (34, 35). However, the restricted application strategies limit its application. In this study, we established a more precise and efficient tool using Cas9n in B. licheniformis to bypass these drawbacks. We tested this system using several original genome editing strategies, including single-gene deletion, double-gene disruption, large DNA fragment deletion, and single-gene integration. Additionally, a host strain capable of high protein production levels, DWc9nΔ7, was constructed using this system.

Bacillus strains are important for the production of various industrial biochemical products, including poly-γ-glutamic acid (36), the broad-spectrum antibiotic bacitracin (37), and 2,3-butanediol (38). However, a lack of tools for genetic manipulation has hindered their development. Thus, it is urgent to find a faster and more efficient genome editing tool to resolve this situation. Although CRISPR-cas9 coupled with the HDR pathway has been used for single-gene knockout and large DNA fragment deletion in Bacillus subtilis (3, 39, 40), genetic manipulation strategies involving Cas9n have rarely been reported in Bacillus, and the genome editing strategies that do exist are limited. However, compared to Cas9-mediated genome editing methods, Cas9n causes less damage to the host and allows for more precise genome editing (4, 31). Based on these advantages, the CRISPR-Cas9n system constructed in this study is a more efficient and systematic method of genome editing. Furthermore, our results will be applicable to other NHEJ-deficient bacterial species.

Based on our results, the CRISPR-Cas9n system constructed in this study can be applied to single-gene deletion using short homology arms (0.1 kb). However, deletion efficiency increased as the size of the homology arms increased, suggesting that larger homology arms improve HDR. Taking into account the electroporation efficiency, 0.3- to 0.5-kb homology arms may be the best choice for single-gene deletion using this system, allowing for deletion efficiencies of up to 80%.

CRISPR-Cas9 has been applied to multiple-gene deletion in human cells and in E. coli (18, 41, 42). However, few reports have focused on the simultaneous deletion of multiple genes using CRISPR-Cas9n in Bacillus. In the present study, it was confirmed that epr and wprA could be simultaneously deleted in B. licheniformis using Cas9n, with a deletion efficiency of 11.6%. The low efficiency of double-gene deletion may have been due to low efficiency of epr deletion. Alternatively, competition between the two sgRNAs in the organism may have led to the differences in efficiency between epr and wprA. Nonetheless, this attempt still has profound significance for the adaptation and development of multiple gene editing methods.

Large DNA fragment deletion using the traditional genome editing method remains problematic. Previously, CRISPR-Cas9 combined with the NHEJ repair pathway has been applied to the deletion of large DNA fragments, ranging from 3 to 17 kb in E. coli with efficiencies of 17.3% to 49.1% (19). Cas9n coupled with dual sgRNAs has been used to delete 36- and 97-kb fragments in E. coli with efficiencies of 20% and 0.7%, respectively (27). In the current study, the bacitracin synthase gene cluster bacABC, a large DNA fragment of 42.7 kb, was completely deleted in one step using a strategy based on CRISPR-Cas9n. The deletion efficiency reached 79.0%, which is much higher than those previously reported. In addition, the sgRNAs used in this strategy targeted different sites of the same DNA strand, whereas previous studies used dual sgRNAs targeted to different DNA strands (40). Furthermore, a gene integration strategy with an efficiency of 76.5% was successfully developed in B. licheniformis via Cas9n. As described in previous research (28), the Cas9n-based integration strategy proved to be an efficient and precise genome insertion tool. Our results also suggest that this strategy could be applied to minimal genome construction in future research.

Bacillus strains have been shown to be suitable hosts for heterologous protein expression. However, abundant extracellular proteases tend to degrade target proteins, limiting target protein production (43–45). Previously, extracellular protease-deficient strains, such as B. subtilis WB600, WB700, and WB800, were constructed to improve heterologous protein production (46, 47). Similarly, we previously constructed the protease-deficient strain B. licheniformis BL10 (mpr, vpr, aprX, epr, bprA, wprA, aprE, bprA, hag, and amyL) to improve nattokinase production and were able to achieve a maximum nattokinase activity level of 33.8 FU/ml (33). In the current study, we deleted six protease genes (epr, wprA, mpr, aprE, vpr, and bprA) and the bacitracin synthase gene cluster bacABC in DWc9n and achieved a maximum nattokinase activity of 59.7 FU/ml in DWc9nΔ7/pPSNT-SsacC, an increase of 25.7% compared to that of the control strain. In this strain, the deletion of bacABC may have led to increases in branched-chain amino acid concentrations, resulting in increased fatty acid accumulation. Thus, the biomass of DWc9nΔ7/pPSNT-SsacC was higher than that of the control strain.

In conclusion, we have developed a systematic genome editing method using Cas9n in B. licheniformis and used this system to successfully perform single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration. Additionally, we constructed a strain, DWc9nΔ7, in which nattokinase activity was increased by 25.7%. This study has described a systematic and efficient genome editing method in B. licheniformis that can be applied to the development of B. licheniformis strains in the future.

MATERIALS AND METHODS

Bacterial strains and culture conditions.The strains and plasmids used in this study are listed in Table 2. B. licheniformis DW2 served as the parent strain for construction of recombinant strains. E. coli DH5α was used to construct recombinant vectors. The plasmid pPHY300PLK was used for construction of sgRNA expression vectors (33). The T2(2)-Ori vector was used to integrate Cas9n (38). All primers used in this study are listed in Table 3.

View this table:
TABLE 2

Strains and plasmids used in this study

View this table:
TABLE 3

Primers used in this study

LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl, pH 7.2) was used for the cultivation of B. licheniformis and E. coli. Antibiotics (20 mg/liter kanamycin, 20 mg/liter tetracycline, and/or 50 mg/liter ampicillin; Sigma-Aldrich) were added to the media as required. B. licheniformis and E. coli were cultivated at 37°C. For nattokinase production, the seed culture was cultivated in a 250-ml flask containing 50 ml LB medium in a rotatory shaker (180 rpm) for 10 h until the optical density at 600 nm (OD600) reached 4.0 to 4.5. Cultures were then transferred (3% inoculum ratio) into nattokinase fermentation medium (2% glucose, 1% peptone, 1% soy peptone, 1.5% yeast extract, 0.5% corn steep liquor, 0.05% K2HPO4·3H2O, 0.01% MgSO4·7H2O, and 0.01% CaCl2·2H2O). The fermentation conditions were the same as those for the seed culture. All of the fermentation experiments were performed in triplicate. Nattokinase activity was detected using the fibrin degradation method as previously described (49).

Construction of the Cas9n expression plasmid.The Cas9n expression plasmid was constructed as previously described (50). Briefly, the P43 promoter from B. subtilis 168 (GenBank accession number K02174.1), the Cas9 nickase gene, and the amyL terminator (amyLt) (FJ556804.1) were amplified using the corresponding primers and fused by splicing by overlap extension (SOE)-PCR. The fused fragments were then inserted into the pHY300PLK vector at the BamHI/XbaI restriction sites. Diagnostic PCR and DNA sequencing were used to confirm that the recombinant plasmid, named pCas9n, was successfully constructed.

Construction of the integration expression plasmid containing the Cas9 nickase gene.The integration expression plasmid containing the gene encoding Cas9 nickase was constructed as previously described (38), and the expression cassette for this gene was integrated into the site of the yjqB gene, which locates in the bacterial phage genome area of B. licheniformis DW2. Since the Cas9 nickase gene expression cluster DNA fragment was too large for gene integration in B. licheniformis, it was divided into two parts, named P1 and P2, that were sequentially integrated into the chromosome of B. licheniformis DW2 (Fig. 1A). First, the P1 gene fragment containing the P43 promoter and part of the cas9 nickase gene was integrated into the chromosome of B. licheniformis DW2 using a previously described protocol (33). The integration was verified using diagnostic PCR and DNA sequencing, and the recombinant strain was named DWcas9n-1. The P2 gene fragment containing the remaining part of the cas9 nickase gene and the amyL terminator then was integrated into the chromosome of DWcas9n-1. Diagnostic PCR and DNA sequencing were used to confirm that the Cas9n expression strain, named DWc9n, was successfully constructed.

Construction of sgRNA expression vectors for genome editing.Four strategies, including single-gene knockout, multiple-gene disruption, large DNA fragment deletion, and single-gene integration, were used to develop a systematic genome editing method. A specific 20-bp protospacer adjacent motif (PAM) sequence was designed using ZiFiT software (http://zifit.partners.org/ZiFiT/), and the sgRNA scaffold was synthesized by splicing using the appropriate primers (Table 3).

The procedure for construction of the sgRNA expression vector for single-gene deletion is shown in Fig. 2A. The ribosome-binding site (RBS)-free P43 promoter coupled with the yvmC sgRNA fragment (see Fig. S2 in the supplemental material), the amyL terminator, and upstream and downstream homology arms of yvmC (1.0 kb) were amplified with the appropriate primers, fused by SOE-PCR, and inserted into the pHY300PLK vector to construct the sgRNA expression plasmid pGRNA-05 (Fig. S3). Meanwhile, to analyze the equilibrium point between the high editing efficiency and sizes of homologous arms, homology arms of 0.1, 0.2, 0.3, 0.5, and 0.7 kb were amplified and inserted into vectors, and the corresponding plasmids were named pGRNA-01, pGRNA-02, pGRNA-03, pGRNA-07, and pGRNA-10, respectively (Fig. S4).

pHY300-ΔeprΔwprA was constructed for simultaneous deletion of the protease genes epr and wprA with the model of p43::gRNA1-p43::gRNA2. Donor templates were spliced together using homology arms (0.3 kb upstream and 0.3 kb downstream) of epr and wprA (Fig. 3A). These fragments then were fused and inserted into the pHY300 vector to construct the sgRNA expression plasmid pHY300-ΔeprΔwprA.

pHY300-ΔbacABC was constructed for deletion of the bacitracin synthetase gene cluster bacABC, a large DNA fragment of 42.7 kb, in B. licheniformis DW2. Two specific sgRNAs were designed to target both sides of the bacABC gene cluster. The donor template contained the DNA sequence 1.0 kb upstream and 1.0 kb downstream of the bacABC gene (Fig. S5). The RBS-free P43 promoter, the sgRNAs, and the homology arms were fused with SOE-PCR, and the fused fragment was inserted into the pHY300PLK vector to construct the sgRNA expression vector pHY300-ΔbacABC.

Construction of the gene integration plasmid was performed using a procedure similar to that of single-gene deletion. Briefly, the RBS-free P43 promoter was coupled with the sgRNA targeting the xkdE site of the phage region in DWc9n, and the aprN gene (FJ374767.1) driven by the P43 promoter was integrated into the chromosome of DWc9n between the 1.0-kb upstream and 1.0-kb downstream homology arms. The fused fragments were inserted into the pHY300PLK vector to generate the aprN expression plasmid pHY300-aprN (Fig. S6).

Determination of editing efficiency.Each of the sgRNA expression plasmids (1 μg) was transformed into the DWc9n host strain by electroporation. Diagnostic PCR was used to verify the transformants, and positive colonies were further verified by DNA sequencing. The gene editing efficiency was calculated as the number of positive colonies as a percentage of the total number of colonies on the plate. At least three transformants were verified for each plasmid.

Plasmid elimination.To eliminate the remaining plasmids in the positive colonies, colonies were cultivated in LB medium without tetracycline at 37°C for several generations, and tetracycline-sensitive colonies were verified with diagnostic PCR.

ACKNOWLEDGMENTS

We are very grateful to Tao Xu (University of Oklahoma, Norman, OK) for providing Cas9 nickase.

This work was funded by the National Program on Key Basic Research Project (973 Program, no. 2015CB150505) and the Science and Technology Program of Wuhan (20160201010086).

FOOTNOTES

    • Received 22 November 2017.
    • Accepted 23 December 2017.
    • Accepted manuscript posted online 12 January 2018.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02608-17.

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KEYWORDS

Bacillus licheniformis
CRISPR-Cas9n
deletion
genome editing
integration
nattokinase production

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