A high-performance gene editing system based on cas12b-guided
By introducing the Cas12b protein and crRNA into Bacillus subtilis, the problems of PAM sequence restriction and the introduction of exogenous resistance genes have been solved, achieving efficient and portable gene editing that is suitable for food industry and synthetic biology research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2024-12-04
- Publication Date
- 2026-06-05
Smart Images
Figure CN122146735A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a high-performance gene editing system based on Cas12b guidance, belonging to the field of genetic engineering technology. Background Technology
[0002] Bacillus subtilis is a Gram-positive strain widely used for exogenous protein expression and is also an ideal chassis microorganism for basic research, thus having wide applications in metabolic engineering and synthetic biology. Currently, Cre / loxP, a widely used genome editing tool in Bacillus subtilis, has been established. However, this type of editing tool introduces exogenous resistance genes as markers for positive selection, which does not meet the requirements of the food industry. Although this system can eliminate resistance genes by introducing a flipper enzyme, the process is cumbersome. Therefore, constructing efficient and portable genome editing tools is of great significance for building higher versions of Bacillus subtilis and using this chassis cell as a microbial cell factory.
[0003] Recently, CRISPR / Cas9-based genome editing tools have been developed in Bacillus subtilis, enabling single-gene deletion. However, the PAM sequences recognized by these systems remain limited, thus restricting their target selection. Therefore, providing a wide-window, rapid, and portable genome editing tool is of great significance for Bacillus subtilis as a metabolic cell factory and synthetic biology research platform. Summary of the Invention
[0004] The present invention provides a gene editing element comprising a Cas12b protein derived from Bacillus hisashii, a crRNA targeting a target gene, and a homologous arm sequence of the target gene; wherein the Cas12b protein and the crRNA are transcribed by different promoters.
[0005] In one embodiment, the gene editing element uses a pHT series plasmid as a vector and contains the gene encoding the Cas12b protein, crRNA targeting the target gene, and the homologous arm sequence of the target gene.
[0006] In one embodiment, the pHT series plasmids include, but are not limited to, pHT01.
[0007] In one implementation, the Cas12b gene is regulated by the P43 promoter.
[0008] In one embodiment, the crRNA targeting the target gene is regulated by the promoter Pveg.
[0009] In one embodiment, the target gene homologous arm sequence is located upstream of the P43 promoter.
[0010] In one embodiment, the homologous arms of the target gene include an upstream homologous arm and a downstream homologous arm; the sequence lengths of the homologous arms are 550–650 bp, respectively.
[0011] In one embodiment, the homologous arms of the target gene also contain the gene sequence to be inserted.
[0012] In one embodiment, the nucleotide sequence of the expression vector pHT-Cas12b-AIO is shown in SEQ ID NO.1.
[0013] In one embodiment, the target gene is a gene expressed by Bacillus subtilis itself, including but not limited to sacA and aprE.
[0014] In one embodiment, the gene editing element contains a Cas12b protein derived from Bacillus hisashii, a crRNA targeting the sacA gene, and a sacA gene homologous arm sequence, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0015] This invention provides a method for gene editing, wherein the gene editing element is transferred into Bacillus subtilis cells.
[0016] In one embodiment, the gene editing includes, but is not limited to, gene knockout or gene insertion.
[0017] In one embodiment, the Bacillus subtilis includes, but is not limited to, Bacillus subtilis 168.
[0018] In one embodiment, the method involves transferring the gene editing element into Bacillus subtilis 168 cells and culturing them in a suitable culture system to achieve the knockout or knock-in of the target gene.
[0019] In one implementation, when knocking out the target gene, the crRNA of the target gene is cloned into the sgRNA position of the sacA gene in the pHT-Cas12b-AIO expression vector, and the upstream and downstream 600bp genes of the target gene are cloned from the Bacillus subtilis 168 genome into the pHT-Cas12b-AIO expression vector to replace the sacA homologous arm. The expression vector is then transformed into Bacillus subtilis 168 and cultured in a culture system to achieve the knockout of the target gene.
[0020] In one embodiment, the crRNA of the target gene is cloned into the sgRNA position of the sacA gene in the pHT-Cas12b-AIO expression vector shown in SEQ ID NO.1, and the upstream homologous arm of the target gene-target gene fragment-downstream homologous arm of the target gene is cloned into the pHT-Cas12b-AIO expression vector to replace the sacA homologous arm. The expression vector is then transformed into Bacillus subtilis 168 and cultured in a culture system to achieve the insertion of the target gene at the target gene position.
[0021] Beneficial effects: This invention first characterized the Cas12b protein from Bacillus hisashii and integrated it into the pHT-AIO-sacA backbone. A low-copy pHT01 vector was selected as the expression vector for crRNA. Using software design, crRNAs targeting different genes were selected and placed downstream of the strongly constitutive promoter Pveg from Bacillus subtilis. Figure 1 The expression vector pHT-Cas12b-AIO was constructed using the sgRNA (as shown in the image). This genome editing method was validated by selecting crRNAs targeting different genes. Colony PCR results showed that both the sacA and aprE genes could be efficiently knocked out.
[0022] The gene editing system and method constructed in this invention have broad application prospects in constructing higher-version chassis cells and in synthetic biology research. Attached Figure Description
[0023] Figure 1 This is a schematic diagram of the construction of the CRISPR-Cas12b gene editing system in B. subtilis.
[0024] Figure 2 A schematic diagram of the CRISPR-Cas12b gene editing system in operation.
[0025] Figure 3 This diagram illustrates the deletion of sacA using the CRISPR-Cas12b gene editing system. M represents the marker; ck represents the control strain B. subtilis 168; and 1-18 represent the knockout strains.
[0026] Figure 4 The deletion of aprE by the CRISPR-Cas12b gene editing system is shown. M stands for Marker; ck represents the control strain B. subtilis 168; 1-10 represent the knockout strains. Detailed Implementation
[0027] LB medium (g / L) -1): Tryptone 10, NaCl 10, yeast extract 5, pH 7.0, add agar powder 20 when preparing solid culture medium.
[0028] Primers and their sequences are shown in Table 1.
[0029] Table 1 Primers required for cloning different crRNAs
[0030]
[0031] P43-BhCas12b expression box:
[0032]
[0033]
[0034] In this context, the single underline indicates the P43 promoter, and the thick underline indicates the rrnB T1 terminator.
[0035]
[0036] The double underlined area represents the Pveg promoter, and the thick underline represents the rrnB T1 terminator.
[0037]
[0038] The double underlined area represents the Pveg promoter, and the thick underline represents the rrnB T1 terminator.
[0039] Transformation method for Bacillus subtilis 168: Inoculate a single colony of Bacillus subtilis 168 into 2 mL of SPI medium and incubate at 37°C with shaking for 12-14 h; Take 100 μL of the culture and inoculate into 5 mL of SPI medium, incubate at 37°C with shaking for 4-5 h, then begin measuring OD. 600 When OD 600 When the concentration reaches approximately 1.0, transfer 200 μL of bacterial culture to 2 mL of SPII medium and incubate at 37 °C and 100 rpm. -1 Incubate on a shaker for 1.5 h; add 20 μL of 100×EGTA solution to the tube and incubate at 37 °C and 100 r·min. -1 After culturing in a shaker for 10 min, aliquot 500 μL into each 1.5 mL centrifuge tube; add an appropriate amount of plasmid (verified by sequencing) to the tube, mix well by pipetting, and incubate at 37 °C and 100 rpm. -1 Incubate in a shaker for 2 hours; after incubation, take about 200 μL of bacterial solution and spread it evenly on the corresponding selective plate, and incubate at 37°C for 12-14 hours.
[0040] Example 1: Construction of the CRISPR-Cas12b system pHT-Cas12b-AIO
[0041] Construction of the Cas12b gene single plasmid editing system: First, the gene was synthesized (by Genewiz, Suzhou, China), and primers were designed. Using primer Cas12b-F / R with pUC-BhCRISPR (SEQ ID NO.4) as a template, the BhCas12b expression cassette (SEQ ID NO.2) was amplified. Using primer Cas12b-bF / R with pHT-AIO-sacA (SEQ ID NO.3) as a template, the corresponding backbone was amplified. The two fragments were then ligated to form pHT-Cas12b. Similarly, using primer BhsgRNA-F / R with pUC-BhCRISPR as a template, BhsgRNA was amplified. Using primer BhsgRNA-bF / R with pHT-Cas12b as a template, the corresponding backbone was amplified. The two fragments were then ligated via homologous recombination to form pHT-Cas12b-AIO.
[0042] The PCR program for the Cas12b gene and pHT01 backbone was as follows: pre-denaturation at 98℃ for 1 min, followed by cycles of denaturation at 98℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for a total of 30 cycles, and a final extension at 72℃ for 10 min. The plasmid template was then removed by digestion with the restriction endonuclease DpnI, and the PCR products were purified. The two fragments were then recombined in vitro using the Infusion recombination method and transformed into *E. coli* JM109 competent cells.
[0043] Example 2: Construction of plasmid pHT-Cas12b-AIO-aprE
[0044] The plasmid pHT-Cas12b-AIO-aprE is an edited plasmid obtained by replacing the target site sacA of plasmid pHT-Cas12b-AIO with the target site aprE. The specific construction steps are as follows:
[0045] A specific sgRNA for BhCas12b was designed on the aprE gene using the CHOPCHOP online software. The crRNA sequence of sacA on pHT-Cas12b-AIO (shown in SEQ ID NO.1) was cloned and replaced using primers PHT-sg aprE-F and PHT-sg aprE-R. The upstream and downstream 600bp genes of the aprE gene were then cloned from the Bacillus subtilis 168 genome into the sacA homologous arm position in the pHT-Cas12b-AIO expression vector using primers pHT-aprE-F and pHT-aprE-F, resulting in the plasmid pHT-Cas12b-AIO-aprE. The expression vector was then transformed into Bacillus subtilis 168 and cultured in a culture system to achieve the knockout or knock-in of the target gene.
[0046] Example 3: Application of the CRISPR-Cas12b system in gene knockout
[0047] The pHT-Cas12b-AIO plasmid constructed in Example 1 and the pHT-Cas12b-AIO-aprE plasmid constructed in Example 2 were transformed into Bacillus subtilis 168, respectively. The resulting recombinant Bacillus subtilis was cultured in LB medium at 37°C and 200 rpm until the OD600 reached 0.4–0.5. Then, it was plated on resistant LB plates to obtain single colonies. Colony PCR was used to determine whether the target gene had been knocked out. Figure 3 and Figure 4 This demonstrates the system's efficiency in single-gene knockout. Knockout efficiency was calculated as the ratio of the number of transformants successfully knocked out the target gene to the number of transformants from randomly selected single colonies on a plate, after colony PCR and sequencing verification.
[0048] like Figure 3 As shown, the sacA gene was knocked out in Bacillus subtilis using pHT-Cas12b-AIO. A total of 18 single colonies were selected for PCR verification, and the sacA gene was knocked out in all 18 colonies, with a knockout efficiency of 100%.
[0049] like Figure 4 As shown, the aprE gene was knocked out in Bacillus subtilis using pHT--Cas12b-AIO-aprE. Ten colonies were selected for PCR verification, and 10 single colonies were found to have knocked out the aprE gene, with a knockout efficiency of 100%.
[0050] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A gene editing element, characterized in that, It contains Cas12b protein, crRNA targeting the target gene, and the target gene homologous arm sequence; the Cas12b protein and the crRNA are transcribed by different promoters.
2. The gene editing element according to claim 1, characterized in that, The gene editing element uses pHT series plasmids as vectors and contains the coding gene for the Cas12b protein, the crRNA targeting the target gene, and the homologous arm sequence of the target gene; the pHT series plasmids include, but are not limited to, pHT01.
3. The gene editing element according to claim 1 or 2, characterized in that, The Cas12b gene is regulated by the P43 promoter; the crRNA targeting the gene is regulated by the Pveg promoter.
4. The gene editing element according to any one of claims 1 to 3, characterized in that, The target gene homologous arm sequence is located upstream of the P43 promoter.
5. The gene editing element according to any one of claims 1 to 4, characterized in that, The target gene also contains the gene sequence to be inserted between its homologous arms.
6. The gene editing element according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.
1.
7. A gene editing method, characterized in that, The gene editing element described in any one of claims 1 to 6 is transferred into Bacillus subtilis cells.
8. The method according to claim 7, characterized in that, The gene editing includes, but is not limited to, gene knockout or gene insertion.
9. The method according to claim 7 or 8, characterized in that, The crRNA of the target gene was cloned into the sgRNA position of the sacA gene in the pHT-Cas12b-AIO expression vector shown in SEQ ID NO.
1. The upstream and downstream homologous arms of the target gene were cloned into the pHT-Cas12b-AIO expression vector to replace the sacA homologous arms. The expression vector was then transformed into Bacillus subtilis 168 and cultured in a culture system to achieve the knockout of the target gene.
10. The method according to claim 7 or 8, characterized in that, The crRNA of the target gene was cloned into the sgRNA position of the sacA gene in the pHT-Cas12b-AIO expression vector shown in SEQ ID NO.
1. The upstream homologous arm of the target gene-target gene fragment-downstream homologous arm of the target gene was cloned into the pHT-Cas12b-AIO expression vector to replace the sacA homologous arm. The expression vector was then transformed into Bacillus subtilis 168 and cultured in a culture system to achieve the insertion of the target gene at the target gene position.