Method for gene editing in intestinal bacteroides based on small cas protein and use thereof
Patent Information
- Application Number
- PCT/CN2025/082416
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2026-09-17
Smart Images
Figure CN2025082416_17092026_PF_FP_ABST
Abstract
Description
A gene editing method and application for Bacteroides oryzae based on a small Cas protein Technical Field
[0001] This invention relates to a gene editing method and application of Bacteroides spp. based on a small Cas protein. Background Technology
[0002] CRISPR / Cas systems have been widely used in gene editing. RNA-guided Cas9 and Cas12a effectors (typically larger than 1000 amino acids) belong to class II and class V CRISPR systems, respectively, and have been widely used as genome editing tools or clinical therapeutics to correct genetic defects. However, these Cas effectors are bulky, making it difficult to efficiently load them into spaced delivery systems with limited capacity, such as bacteriophages or adeno-associated viruses (AAVs).
[0003] Reports indicate that the small CRISPR effectors CasX (Cas12e, 986 amino acids) and CasΦ (Cas12j, 700-800 amino acids) have been used as functional genome editors in *E. coli* and human cells. The Cas12f system, with its smaller Cas effector protein (400-700 amino acids), shares sequence similarity with the transposon-encoded TnpB nuclease. In 2020, Virginijus Siksnys' team demonstrated that Cas12f can perform PAM-dependent double-stranded DNA cleavage in vitro. In 2021, Yong-Sam Kim's team, Qi Lei's team, and Ji Quanjiang's team independently demonstrated the mammalian cell and bacterial editing activities of Un1Cas12f1 (537 amino acids, recognizing TTT PAM) and AsCas12f1 (422 amino acids, recognizing TTR PAM). To elucidate the molecular mechanism by which such a small AsCas12f1 nuclease possesses editing activity and to enhance its editing activity, Ji Quanjiang's team constructed the AsCas12f1-sgRNA-dsDNA ternary complex structure in 2023. Based on the structure, they conducted rational design, which greatly improved its gene editing efficiency.
[0004] In the enteric bacterium *Bacteroides polymorpha*, Timothy K. Lu et al. proposed a CRISPR / dCas9-based technique to inhibit the expression of some genes. This system, guided by gRNA, successfully inhibited the expression of the *Bacteroides polymorpha* Bt1754 and Bt1854 genes. The CRISPR / dCas9 technology is based on the principle of dCas9 inhibiting gene transcription, thereby achieving gene expression downregulation. A drawback of this technique is that it can only achieve gene expression downregulation.
[0005] In the *Bacteroides* genus *Pleurotus*, Justin L. Sonnenburg et al. achieved the insertion of a 70kbp exogenous fragment using pNBU2 technology. In the *Bacteroides* genus, Andrew L. Goodman et al. achieved single-gene deletion in *Bacteroides* through homologous double crossover using the pExchange-tdk suicide plasmid. NBU1 or NBU2 technologies are based on integrase and integration sites, inserting all elements of the exogenous plasmid (including selection markers such as antibiotic genes) into a specific site in the genome under the action of integrase. A drawback of this technology is that it cannot produce mutant strains without selection markers. The pExchange-tdk suicide plasmid technology requires the prior construction of a gene lacking the tdk gene, involving multiple steps and a large workload, which limits its application in the research and modification of *Bacteroides* bacteria.
[0006] Dai Lei's team constructed Bacteroides gene editing tools based on CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-SpRY, with Cas proteins ranging in size from 1300 to 1400 amino acids. However, further integration into the phage genome (the difficulty of integrating foreign genes into the phage genome is directly proportional to the size of the foreign gene) presents objective difficulties for achieving in situ gene editing of microbial communities via phage delivery, and plasmid construction is time-consuming and labor-intensive.
[0007] Existing gene editing technologies for the gut microbiota Bacteroides suffer from drawbacks such as redundant operational steps, cumbersome plasmid construction, and large amino acid volumes of effector proteins. These shortcomings pose technical obstacles to the research and application of Bacteroides. Summary of the Invention
[0008] One object of the present invention is to provide a gene editing method for Bacteroides spp., an intestinal bacterium, based on a small Cas protein.
[0009] On one hand, this invention provides a gene editing method for the enteric bacterium Bacteroides based on a small Cas protein. This method primarily utilizes a small Cas protein to construct a small, efficient, and traceless editing system within the genus Bacteroides to achieve gene editing. The gene editing method of this invention includes: constructing a CRISPR / Cas editing system using a replicative plasmid vector to edit the target gene in Bacteroides; wherein the constructed CRISPR / Cas editing system is a CRISPR / Cas12f, CRISPR / CasΦ, or CRISPR / TnpB system.
[0010] Specifically, this invention provides a gene editing method for Bacteroides spp., an enteric bacterium, based on a small Cas protein. The method includes:
[0011] Constructing the target plasmid: Construct a target plasmid including pB025-backbone, inducible promoter, Cas protein gene, gRNA, and upstream and downstream homologous arms of the target gene; the Cas protein gene is Cas12f, CasΦ, or TnpB, and the length of the upstream and downstream homologous arms of the target gene is 500-1000bp;
[0012] Transformation of Escherichia coli: The constructed target plasmid was transformed into competent Escherichia coli cells and screened in a medium containing the first selection marker to obtain Escherichia coli containing the target plasmid;
[0013] Conjugation and screening: Escherichia coli containing the target plasmid and wild-type Bacteroides were cultured to the logarithmic phase, and then screened to obtain Bacteroides containing the target plasmid.
[0014] Gene editing was performed by culturing Bacteroides containing the target plasmid in a liquid medium containing a second selection marker and an inducer to the logarithmic phase, resulting in Bacteroides mutants that had undergone editing and contained antibiotic resistance markers.
[0015] In the above gene editing method, preferably, the Cas protein gene is Cas12f.
[0016] In the gene editing methods described above, the constructed CRISPR / Cas editing system uses an inducible promoter to express the Cas protein.
[0017] In the above gene editing methods, the Bacteroides gene editing method based on the small Cas protein further includes:
[0018] Loss of plasmids during passage: The edited Bacteroides mutant strain containing antibiotic resistance markers was added to a medium without selection markers and passaged for 1-15 generations. The resulting strain was then diluted and plated on a plate without selection markers to obtain Bacteroides mutant strains without selection markers and without exogenous plasmids.
[0019] In the gene editing methods described above, the chemically transformed E. coli is S17.
[0020] In the above gene editing method, preferably, the process of transforming Escherichia coli includes: mixing the successfully assembled plasmid with Escherichia coli S17 competent cells, placing them on ice in an ice bath, then performing heat shock, cooling them on ice, adding antibiotic-free liquid culture medium, culturing them in an incubator, activating them, and then spreading the bacterial solution on a plate containing a first selection marker.
[0021] In the above gene editing method, the length of the upstream and downstream homologous arms of the target gene is 500-1000bp, for example, it can be 500bp, 600bp, 750bp, 800, 850bp, 900bp, 1000bp, preferably 500bp.
[0022] In the above gene editing method, the process of conjugation and screening includes: culturing Escherichia coli containing plasmids and wild-type Bacteroides to the logarithmic phase, mixing the two bacteria, centrifuging, discarding the supernatant, suspending the bacterial mixture precipitate in liquid culture medium, and then transferring it to a plate without selection markers for aerobic culture; then scraping the bacterial growth, eluting it in liquid culture medium, taking a portion of the liquid and placing it on a solid plate containing a second selection marker, spreading it evenly, and culturing it under anaerobic conditions.
[0023] In the above gene editing method, the process includes: culturing Bacteroides containing the target plasmid in a liquid culture medium containing a second selection marker and an inducer until it reaches the logarithmic growth phase, then performing serial dilutions, and then plating it onto a solid plate containing the second selection marker and an inducer, and culturing it under anaerobic conditions; then picking single clones from the selection plate and culturing them overnight in a culture medium containing the second selection marker; and then identifying whether the target gene has been edited.
[0024] In the above gene editing method, the first screening marker includes an ampicillin resistance marker;
[0025] Preferably, the second screening marker includes an erythromycin resistance marker and a gentamicin resistance marker;
[0026] Preferably, the inducing agent includes an aTc inducing agent.
[0027] In the above gene editing method, the process of losing plasmids through passage includes: adding liquid culture medium without selection markers to the Bacteroides mutant strain that has been edited and contains selection marker resistance, or streaking it on a plate without selection markers, and passaged it continuously for 10-15 generations to obtain Bacteroides mutant strains without selection markers and without exogenous plasmids.
[0028] In some specific embodiments of this invention, the editing efficiency of three CRISPR / Cas systems—CRISPR / Cas12f, CRISPR / CasΦ (CasΦ2), and CRISPR / TnpB (ISDge10)—in Bacteroides fragilis was compared. Specifically, in Bacteroides fragilis, different gRNAs were designed for the gene BF9343_RS15240 corresponding to different CRISPR / Cas systems. The results showed that CRISPR / Cas12f had the highest efficiency among the three CRISPR / Cas systems.
[0029] In some specific embodiments of the present invention, the editing effects of the CRISPR / AsCas12f system in three types of Bacteroides: Bacteroides polymorpha (Bt), Bacteroides fragilis (Bf), and Bacteroides vulgaris (Bv) were compared. Specifically, using CRISPR / AsCas12f, different gRNAs were designed for the asparaginase gene in different Bacteroides species. The results showed that CRISPR / Cas12f could achieve gene editing in all three types of Bacteroides, with the highest efficiency in Bacteroides polymorpha (Bt) and Bacteroides fragilis (Bf).
[0030] In some specific embodiments of the present invention, the effects of homologous arms of different lengths on the gene editing method provided by the present invention were compared. Specifically, the present invention designed repair templates with homologous arms of different lengths for transformation experiments, using CRISPR / AsCas12f, targeting the asparaginase gene, and shortened the homologous arm from 1000 bp to 500, 250, and 100 bp in Bacteroides fragilis, respectively. The results showed that the editing efficiency of the 500 bp homologous arm was 94.7% (18 / 19), and the editing efficiency of the 250 bp homologous arm was 4.5% (1 / 22). The 500 bp homologous arm can be considered as the optimal length for a small CRISPR / AsCas12f editing system.
[0031] In some specific embodiments of the present invention, the gRNA design can be carried out with reference to existing technologies. For example, the CHOPCHOP online tool can be used, the specific URL of which is: http: / / chopchop.cbu.uib.no / .
[0032] According to a specific embodiment of the present invention, in the gene editing method of Bacteroides in the intestinal bacteria based on small Cas protein of the present invention, the intestinal Bacteroides includes, but is not limited to, Bacteroides polymorpha, Bacteroides fragilis, Bacteroides commonis, Bacteroides ovalis, and Bacteroides regularis.
[0033] This invention can be applied to bacterial species within the genus Bacteroides, and can also be used for gene editing with Bacteroides bacteriophages, including the deletion and insertion of large gene clusters.
[0034] On the other hand, the present invention also provides mutant strains obtained by the Bacteroides enteroides gene editing method based on the small Cas protein according to the present invention, which have large deletions and / or insertions of gene clusters compared with wild type.
[0035] In summary, this invention constructs a gene editing method for *Bacteroides* based on a small Cas protein using the CRISPR / Cas system. It also investigates the impact of homologous arm length on editing efficiency, determining an optimal range for its length. Single-gene and gene cluster knockout and insertion were achieved in *Bacteroides*, realizing a streamlined gene editing process. Single-gene knockout efficiency was improved, yielding marker-free mutant strains. The CRISPR / Cas12f editing system provided by this invention is smaller in size, has more potential applications, and is simpler and faster to operate. Attached Figure Description
[0036] Figure 1 is a schematic diagram of the process of gene editing in Bacteroides using the CRISPR / Cas gene editing system of the present invention.
[0037] Figure 2 shows the structure of the plasmid obtained in Example 1.
[0038] Figure 3 shows the structure of the plasmid obtained in Example 1.
[0039] Figure 4 shows the structure of the plasmid obtained in Example 1.
[0040] Figure 5 is a schematic diagram of the cleavage mechanism of three small Cas proteins in Example 1 of the present invention.
[0041] Figure 6 is a map of the integrated plasmid for gene editing in Bacteroides fragilis in Example 1 of this invention.
[0042] Figure 7 shows the PCR verification results of knocking out the BF9343_RS15240 gene in Bacteroides fragilis in Example 1 of this invention.
[0043] Figure 8 shows the results of three small Cas gene editing methods in Bacteroides fragilis in Example 1 of this invention.
[0044] Figure 9 shows the growth phenotype test results of wild-type and mutant strains of Bacteroides fragilis in Example 1 of the present invention with fructose as the sole carbon source.
[0045] Figure 10 is a schematic diagram of the CRISPR / AsCas12f gene editing tool targeting and knocking out genes (asparaginase) in Example 2 of the present invention. Two gRNAs were designed for Bf and Bv, and five gRNAs were designed for Bt.
[0046] Figure 11 shows the editing results of the CRISPR / AsCas12f gene editing tool on three Bacteroides species in Example 2 of this invention.
[0047] Figure 12 shows the PCR verification results of the CRISPR / AsCas12f gene editing tool in knocking out the asparaginase gene in three Bacteroides species in Example 2 of this invention.
[0048] Figure 13 is a schematic diagram of the construction of plasmids with different homologous arm sizes in Example 3 of the present invention.
[0049] Figure 14 shows the editing results of homologous arms of different lengths in Embodiment 3 of the present invention.
[0050] Figure 15 shows the PCR verification of transformants with different homologous arms in Example 3 of the present invention. Detailed Implementation
[0051] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the essence of the invention are within the scope of the invention.
[0052] In all embodiments, unless otherwise specified, all materials and biochemical reagents used are commercially available. Primers, Cas12f gene fragments, and other components were synthesized by third-party companies. Unless otherwise specified, the techniques used are conventional methods well-known to those skilled in the art, or were performed according to the conditions recommended by the instrument manufacturer.
[0053] Unless otherwise specified, all sequences described in this invention are in the 5'-3' direction.
[0054] Example 1: Deletion of the fructose metabolism regulating gene BF RS15240 in Bacteroides fragilis and comparison of editing efficiency of different Cas systems.
[0055] In this embodiment, the gene editing process in Bacteroides is shown in Figure 1. It mainly includes: plasmid construction, chemical transformation of S17, conjugation of S17 containing the target plasmid into wild-type Bacteroides, and erythromycin screening to obtain wild-type Bacteroides containing the target plasmid, culturing and inducing to obtain mutant strains containing the target plasmid, and subculturing. The specific operations are as follows:
[0056] (1) gRNA design:
[0057] The gRNA design in this patent uses the CHOPCHOP online tool, the specific URL of which is: http: / / chopchop.cbu.uib.no / .
[0058] (2) Plasmid design:
[0059] The plasmid designed in this patent mainly consists of four parts: the pB025 plasmid backbone, the Cas12f, CasΦ, or TnpB protein gene, gRNA, and homologous arms. This series of elements is assembled into a single plasmid primarily using the Gibson assembly method. Specific information about each element is as follows:
[0060] ① First, linearize the pB025 plasmid backbone, Cas12f element (which can also be replaced with CasΦ or TnpB), and the upstream and downstream homologous arms of the target gene BF RS15240. Linearization will result in 20-25 bp repetitive sequences between elements (this needs to be considered before primer design). Then, assemble the primers in a 10 μL volume, containing 0.05 pmol of the pB025 plasmid backbone, 0.1 pmol of the Cas12f element, 0.1 pmol each of the upstream and downstream homologous arms of the target gene, and 5 μL of 2× [presumably a specific primer design unit]. HiFi DNA Assembly Master Mix (NEB, E2621S) was added to a final volume of 10 μL with water. The mixture was then placed in a PCR instrument and incubated at 50°C for 1 hour. The assembled product was then transduced into S17 E. coli competent cells (Beijing Zhuangmeng). The plasmid p134-TetR-Ptet-Cas12f-HA-HB was obtained by screening on LB ampicillin plates.
[0061] ② The obtained plasmid p134-TetR-Ptet-Cas12f-HA-HB was goldenized using BspQ1 restriction enzyme and T4 ligase. Add 20 ng of p134-TetR-Ptet-Cas12f-HA-HB plasmid, 1 μL of BspQ1 restriction enzyme (NEB, R0712S), 1 μL of T4 ligase (NEB, M0202), 1 μL of annealed gRNA product, and 1 μL of 10×T4 buffer (NEB, M0202) to a 10 μL system, and then add water to bring the volume to 10 μL. Cycle 6 times: 37℃ for 5 min, 25℃ for 5 min, 37℃ for 10 min, 65℃ for 5 min, and store at 4℃. The other methods are the same as above, and the p134-TetR-Ptet-Cas12f-HA-HB-gRNA plasmid can be obtained. The plasmid structure is shown in Figure 2. Figures 3 and 4 are the plasmid structures obtained by replacing the Cas12f element with TnpB (ISDge10) and CasΦ (CasΦ2), respectively. The specific DNA base sequence information of the elements is shown in Table 1.
[0062] (3) Chemical transformation of S17: Take 5 μL of the successfully assembled plasmid and add it to 100 μL of S17 competent cells. Place it on ice for 30 minutes, heat shock it at 42℃ for 90 seconds, place it on ice for 5 minutes, add 500 μL of LB liquid medium, and then place it in a 37℃ incubator at 200 rpm for 1 hour. Finally, spread it on an LB solid plate containing ampicillin resistance.
[0063] (4) Conjugation and screening of wild-type Bacteroides: S17 Escherichia coli containing plasmids and wild-type Bacteroides were cultured to the logarithmic phase, so that the OD600 of the two strains was between 0.4 and 0.6. They were mixed at a volume ratio of 1:1, centrifuged at 5000 rpm for 20 minutes, the supernatant was discarded, and the bacterial mixture was resuspended in 100 μL of BHI liquid medium. It was then transferred to antibiotic-free BHI-AGAR plates and cultured at 37°C under aerobic conditions for 24 hours. The bacterial growth was scraped off with a cotton swab, washed into 1 mL of BHI liquid medium, and 150 μL was transferred to BHI-AGAR plates containing erythromycin and gentamicin (the plates contained 100 μg / mL gentamicin and 25 μg / mL erythromycin). The plates were spread evenly and cultured in an anaerobic incubator at 37°C for 2 days.
[0064] (5) Bacteroidetes gene editing:
[0065] ① After Bacteroides conjugation and screening on BHI-AGAR plates containing erythromycin and gentamicin (the plates contain 100 μg / ml gentamicin and 25 μg / ml erythromycin), single clones were selected and cultured in BHI liquid medium (containing 100 μg / ml gentamicin and 25 μg / ml erythromycin). Then, PCR identification was performed using specific primers to determine whether the single clone contained the target plasmid.
[0066] ② Culture Bacteroides containing the target plasmid in BHI liquid medium (containing 100 μg / ml gentamicin, 25 μg / ml erythromycin, and 100 ng / ml aTc inducer) at a volume ratio of 1%, and incubate at 37°C under anaerobic conditions for approximately 15 hours. Perform serial dilutions (1-fold, 10-fold, 100-fold, and 1000-fold dilutions), and spread 100 μL of each dilution onto BHI-AGAR plates (containing 100 μg / ml gentamicin, 25 μg / ml erythromycin, and 100 ng / ml aTc inducer). Incubate at 37°C under anaerobic conditions for approximately 40-48 hours.
[0067] ③ Select single colonies grown on the plate and culture them in BHI liquid medium (containing 100 μg / ml gentamicin and 25 μg / ml erythromycin) overnight under anaerobic conditions at 37°C. Use designed primers to perform PCR to identify whether gene editing has occurred in the bacterial culture. Specifically: First, the primers used for identification are designed outside the homologous arms of the genome; second, if a gene deletion experiment is performed, the PCR band after identification will be smaller than that of the wild type, and the PCR product can be sent for sequencing confirmation; third, if a gene insertion experiment is performed, the PCR band after identification will be larger than that of the wild type, and the PCR product can be sent for sequencing confirmation.
[0068] (6) Bacteroidetes passage:
[0069] The purpose of Bacteroides passage is to obtain mutant strains with a clean background and no selection markers, which is of great significance for the engineering of live Bacteroides drugs. Specifically, mutant strains confirmed to have undergone gene editing are inoculated at a volume ratio of 0.5% into antibiotic-free BHI liquid medium and passaged for 10-15 generations. The bacterial culture is then serially diluted (1-fold, 10-fold, 100-fold) and plated on antibiotic-free BHI-AGAR plates. The plates are then anaerobic at 37°C, and amplified using specific primers attached to the plasmid to determine if the plasmid has been lost. If the plasmid is lost, no band is produced after PCR amplification with the specific primers; if the plasmid is present, a band is produced after PCR amplification with the specific primers.
[0070] Table 1. DNA base sequence information for specific elements.
[0071] Figures 5-9 show the construction of three small CRISPR / Cas gene editing systems in *Bacteroides fragilis* according to this invention. Figure 5 shows a schematic diagram of the cleavage mechanism of the three small Cas proteins. Figure 6 shows an integrated plasmid map of gene editing. Figure 7 shows the PCR verification results of knocking out the BF9343_RS15240 gene. Figure 8 shows the gene editing results of the three small Cas proteins. Figure 9 shows the growth phenotype tests of wild-type and mutant strains using fructose as the sole carbon source.
[0072] In summary, this invention enables the deletion of fructose metabolism regulatory genes in Bacteroides fragilis, and successfully achieved the deletion of fructose metabolism genes using the technical solution of this invention. The editing efficiency of three CRISPR / Cas systems—CRISPR / Cas12f, CRISPR / CasΦ (CasΦ2), and CRISPR / TnpB (ISDge10)—was compared, with CRISPR / Cas12f exhibiting the highest efficiency.
[0073] Example 2: Knockout of the asparagine gene, a gene involved in nitrogen source utilization, in Bacteroides polymorpha (Bt), Bacteroides fragilis (Bf), and Bacteroides vulgaris (Bv).
[0074] In this embodiment, the gene editing process in Bacteroides is shown in Figure 1. It mainly includes: plasmid construction, chemical transformation of S17, conjugation of S17 containing the target plasmid into wild-type Bacteroides, and erythromycin screening to obtain wild-type Bacteroides containing the target plasmid, culturing and inducing to obtain mutant strains containing the target plasmid, and subculturing. The specific operations are as follows:
[0075] (1) gRNA design:
[0076] The procedure was as described in Example 1, using Cas12f to target and knock out the asparagine gene to design two gRNAs for Bf and Bv, and five gRNAs for Bt.
[0077] (2) Plasmid design:
[0078] The plasmid designed in this patent mainly consists of three parts: the p134-TetR-Ptet-Cas12f-HA-HB plasmid obtained in Example 1, the asparagine upstream and downstream homologous arms, and gRNA. The Gibson assembly method is primarily used to assemble this series of elements into a single plasmid. Specific information regarding each element is as follows:
[0079] ① First, linearize the plasmid p134-TetR-Ptet-Cas12f-HA-HB and the upstream and downstream homologous arms of asparagine from Example 1. After linearization, there will be 20-25 bp repetitive sequences between each element (this needs to be considered before designing primers). Then, assemble the plasmid in a 10 μL system, which contains 0.05 pmol of plasmid backbone, 0.1 pmol each of the upstream and downstream homologous arms of the target gene, 5 μL of 2×NEBuilder HiFi Assembly Mix, and water to make up to 10 μL. Then, place it in a PCR instrument and react at 50℃ for 1 h. After that, transform the assembly product into S17 E. coli competent cells and screen on LB ampicillin plates to obtain plasmid p134-TetR-Ptet-Cas12f-asparagineHAB.
[0080] ② The obtained plasmid p134-TetR-Ptet-Cas12f-asparagineHAB was assembled using BspQ1 restriction enzyme and T4 ligase via Goldengate. Add 20 ng of p134-TetR-Ptet-Cas12f-asparagineHAB plasmid, 1 μL of BspQ1 restriction enzyme, 1 μL of T4 ligase, 1 μL of annealed gRNA product, and 1 μL of 10×T4 buffer to a 10 μL system, and then add water to bring the volume to 10 μL. Cycle at 37℃ for 5 min, 25℃ for 5 min, 6 times; then at 37℃ for 10 min, 65℃ for 5 min, and store at 4℃. Repeat the above steps to obtain the p134-TetR-Ptet-Cas12f-asparagineHAB-gRNA plasmid.
[0081] (3) Chemical transformation of S17: Take 5 μL of the successfully assembled plasmid and add it to 100 μL of S17 competent cells. Place it on ice for 30 minutes, heat shock it at 42℃ for 90 seconds, place it on ice for 5 minutes, add 500 μL of LB liquid medium, and then place it in a 37℃ incubator at 200 rpm for 1 hour. Finally, spread it on an LB solid plate containing ampicillin resistance.
[0082] (4) Conjugation and screening of wild-type Bacteroides: S17 Escherichia coli containing plasmids and wild-type Bacteroides were cultured to the logarithmic phase, so that the OD600 of the two strains was between 0.4 and 0.6. They were mixed at a volume ratio of 1:1, centrifuged at 5000 rpm for 20 minutes, the supernatant was discarded, and the bacterial mixture was resuspended in 100 μL of BHI liquid medium. It was then transferred to antibiotic-free BHI-AGAR plates and cultured at 37°C under aerobic conditions for 24 hours. The bacterial growth was scraped off with a cotton swab, washed into 1 mL of BHI liquid medium, and 150 μL was transferred to BHI-AGAR plates containing erythromycin and gentamicin (the plates contained 100 μg / mL gentamicin and 25 μg / mL erythromycin). The plates were spread evenly and cultured in an anaerobic incubator at 37°C for 2 days.
[0083] (5) Bacteroidetes gene editing:
[0084] ① After Bacteroides conjugation and screening on BHI-AGAR plates containing erythromycin and gentamicin (the plates contain 100 μg / ml gentamicin and 25 μg / ml erythromycin), single clones were selected and cultured in BHI liquid medium (containing 100 μg / ml gentamicin and 25 μg / ml erythromycin). Then, PCR identification was performed using specific primers to determine whether the single clone contained the target plasmid.
[0085] ② Culture Bacteroides containing the target plasmid in BHI liquid medium (containing 100 μg / ml gentamicin, 25 μg / ml erythromycin, and 100 ng / ml aTc inducer) at a volume ratio of 1%, and incubate at 37°C under anaerobic conditions for approximately 15 hours. Perform serial dilutions (1-fold, 10-fold, 100-fold, and 1000-fold dilutions), and spread 100 μL of each dilution onto BHI-AGAR plates (containing 100 μg / ml gentamicin, 25 μg / ml erythromycin, and 100 ng / ml aTc inducer). Incubate at 37°C under anaerobic conditions for approximately 40-48 hours.
[0086] ③ Select single colonies grown on the plate and culture them in BHI liquid medium (containing 100 μg / ml gentamicin and 25 μg / ml erythromycin) overnight under anaerobic conditions at 37°C. Use designed primers to perform PCR to identify whether gene editing has occurred in the bacterial culture. Specifically: First, the primers used for identification are designed outside the homologous arms of the genome; second, if a gene deletion experiment is performed, the PCR band after identification will be smaller than that of the wild type, and the PCR product can be sent for sequencing confirmation; third, if a gene insertion experiment is performed, the PCR band after identification will be larger than that of the wild type, and the PCR product can be sent for sequencing confirmation.
[0087] (6) Bacteroidetes passage:
[0088] The purpose of Bacteroides passage is to obtain mutant strains with a clean background and no selection markers, which is of great significance for the engineering of live Bacteroides drugs. Specifically, mutant strains confirmed to have undergone gene editing are inoculated at a volume ratio of 0.5% into antibiotic-free BHI liquid medium and passaged for 10-15 generations. The bacterial culture is then serially diluted (1-fold, 10-fold, 100-fold) and plated on antibiotic-free BHI-AGAR plates. The plates are then anaerobic at 37°C, and amplified using specific primers attached to the plasmid to determine if the plasmid has been lost. If the plasmid is lost, no band is produced after PCR amplification with the specific primers; if the plasmid is present, a band is produced after PCR amplification with the specific primers.
[0089] Figures 10-12 show the editing effects of the CRISPR / AsCas12f gene editing tool on other Bacteroides species. Figure 10 shows a schematic diagram of the targeted gene knockout (asparaginase), with two gRNAs designed for Bf and Bv respectively, and five gRNAs designed for Bt. Figure 11 shows the editing results of the CRISPR / AsCas12f gene editing tool in the three Bacteroides species. Figure 12 shows the PCR verification results of the CRISPR / AsCas12f gene editing tool in knocking out the asparaginase gene in the three Bacteroides species. The results show that CRISPR / Cas12f can achieve gene editing in all three Bacteroides species, with the highest efficiency in Bacteroides polymorpha (Bt) and Bacteroides fragilis (Bf). The sequences of each element are shown in Table 2.
[0090] Table 2
[0091] Example 3 explores the length range of homologous arms in the CRISPR / AsCas12f editing system.
[0092] This invention investigates the impact of homologous arm lengths on gene editing through transformation experiments using repair templates with different homologous arm lengths. Using the plasmid vector and homologous arms described above, the homologous arm length of BF_ansB (asparagine from Bacteroides fragilis (Bf)) was shortened from 1000 bp to 500, 250, and 100 bp (as shown in Figure 13). The results showed that the 1000 bp homologous arm had 100% (22 / 22) editing efficiency, and the 500 bp homologous arm had 94.7% (18 / 19). However, when the homologous arm length was reduced to 250 bp, the editing efficiency was only 4.5% (1 / 22), and at 100 bp, the editing efficiency dropped to 0 (0 / 22) (as shown in Figures 14 and 15). These results indicate that the homologous arm length has a significant impact on the editing efficiency of the small CRISPR / AsCas12f editing system, and 500 bp homologous arms can be considered the optimal length for the small CRISPR / AsCas12f editing system. The specific steps are as follows:
[0093] (1) Construction of plasmid vectors with homologous arms of different sizes:
[0094] ① Design primers to linearize the backbone and homologous arms (HAB) of the p134-TetR-Ptet-Cas12f-asparagineHAB-gRNA11 plasmid, amplifying the upstream and downstream homologous arms to 500bp, 250bp, and 100bp respectively. Linearization will result in 20-25bp repetitive sequences between elements (this needs to be considered before primer design). Then, assemble the plasmid in a 10 μL volume, containing 0.05 pmol of the plasmid backbone, 0.1 pmol of the target gene homologous arm fragment, 5 μL of 2×NEBuilder HiFi Assembly Mix, and add water to a final volume of 10 μL. Incubate at 50°C for 1 h. Transform the assembled product into S17 E. coli competent cells and screen on LB ampicillin plates to obtain the plasmid p134-TetR-Ptet-Cas12f-500bp. The three plasmids are asparagineHAB-gRNA11, p134-TetR-Ptet-Cas12f-250bp asparagineHAB-gRNA11, and p134-TetR-Ptet-Cas12f-100bp asparagineHAB-gRNA11.
[0095] (2) Chemical transformation of S17: Take 5 μL of the successfully assembled plasmid and add it to 100 μL of S17 competent cells. Place it on ice for 30 minutes, heat shock it at 42℃ for 90 seconds, place it on ice for 5 minutes, add 500 μL of LB liquid medium, and then place it in a 37℃ incubator at 200 rpm for 1 hour. Finally, spread it on an LB solid plate containing ampicillin resistance.
[0096] (3) Conjugation and screening of wild-type Bacteroides: S17 Escherichia coli containing plasmids and wild-type Bacteroides were cultured to the logarithmic phase, so that the OD600 of the two strains was between 0.4 and 0.6. They were mixed at a volume ratio of 1:1, centrifuged at 5000 rpm for 20 minutes, the supernatant was discarded, and the bacterial mixture was resuspended in 100 μL of BHI liquid medium. It was then transferred to antibiotic-free BHI-AGAR plates and cultured at 37°C under aerobic conditions for 24 hours. The bacterial growth was scraped off with a cotton swab, washed into 1 mL of BHI liquid medium, and 150 μL was transferred to BHI-AGAR plates containing erythromycin and gentamicin (the plates contained 100 μg / mL gentamicin and 25 μg / mL erythromycin). The plates were spread evenly and cultured in an anaerobic incubator at 37°C for 2 days.
[0097] (4) Bacteroidetes gene editing:
[0098] ① After Bacteroides conjugation and screening on BHI-AGAR plates containing erythromycin and gentamicin (the plates contain 100 μg / ml gentamicin and 25 μg / ml erythromycin), single clones were selected and cultured in BHI liquid medium (containing 100 μg / ml gentamicin and 25 μg / ml erythromycin). Then, PCR identification was performed using specific primers to determine whether the single clone contained the target plasmid.
[0099] ② Culture Bacteroides containing the target plasmid in BHI liquid medium (containing 100 μg / ml gentamicin, 25 μg / ml erythromycin, and 100 ng / ml aTc inducer) at a volume ratio of 1%, and incubate at 37°C under anaerobic conditions for approximately 15 hours. Perform serial dilutions (1-fold, 10-fold, 100-fold, and 1000-fold dilutions), and spread 100 μL of each dilution onto BHI-AGAR plates (containing 100 μg / ml gentamicin, 25 μg / ml erythromycin, and 100 ng / ml aTc inducer). Incubate at 37°C under anaerobic conditions for approximately 40-48 hours.
[0100] ③ Select single colonies grown on the plate and culture them in BHI liquid medium (containing 100 μg / ml gentamicin and 25 μg / ml erythromycin) overnight under anaerobic conditions at 37°C. Use designed primers to perform PCR to identify whether gene editing has occurred in the bacterial culture. Specifically: First, the primers used for identification are designed outside the homologous arms of the genome; second, if a gene deletion experiment is performed, the PCR band after identification will be smaller than that of the wild type, and the PCR product can be sent for sequencing confirmation; third, if a gene insertion experiment is performed, the PCR band after identification will be larger than that of the wild type, and the PCR product can be sent for sequencing confirmation.
[0101] (5) Bacteroidetes passage:
[0102] The purpose of Bacteroides passage is to obtain mutant strains with a clean background and no selection markers, which is of great significance for the engineering of live Bacteroides drugs. Specifically, mutant strains confirmed to have undergone gene editing are inoculated at a volume ratio of 0.5% into antibiotic-free BHI liquid medium and passaged for 10-15 generations. The bacterial culture is then serially diluted (1-fold, 10-fold, 100-fold) and plated on antibiotic-free BHI-AGAR plates. The plates are then anaerobic at 37°C, and amplified using specific primers attached to the plasmid to determine if the plasmid has been lost. If the plasmid is lost, no band is produced after PCR amplification with the specific primers; if the plasmid is present, a band is produced after PCR amplification with the specific primers.
[0103] The sgRNAs used in this invention are shown in Table 3.
[0104] Table 3
[0105] The primers used in this invention are shown in Table 4.
[0106] Table 4
[0107] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention to achieve the same purpose. Therefore, all equivalent technical solutions also fall within the protection scope of this invention.
Claims
1. A gene editing method for Bacteroides oryzae based on a small Cas protein, the method comprising: Constructing the target plasmid: Construct a target plasmid including pB025-backbone, inducible promoter, Cas protein gene, gRNA, and upstream and downstream homologous arms of the target gene; the Cas protein gene is Cas12f, CasΦ, or TnpB, and the length of the upstream and downstream homologous arms of the target gene is 500-1000bp; Transformation of Escherichia coli: The constructed target plasmid was transformed into competent Escherichia coli cells and screened in a medium containing the first selection marker to obtain Escherichia coli containing the target plasmid; Conjugation and screening: Escherichia coli containing the target plasmid and wild-type Bacteroides were cultured to the logarithmic phase, and then screened to obtain Bacteroides containing the target plasmid. Gene editing was performed by culturing Bacteroides containing the target plasmid in a liquid medium containing a second selection marker and an inducer to the logarithmic phase, resulting in Bacteroides mutants that had undergone editing and contained antibiotic resistance markers.
2. The gene editing method of claim 1, wherein, In the constructed CRISPR / Cas editing system, an inducible promoter is used to express the Cas protein.
3. The gene editing method according to claim 1, further comprising: Loss of plasmids during passage: The edited Bacteroides mutant strain containing antibiotic resistance markers was added to a medium without selection markers and passaged for 1-15 generations. The resulting strain was then diluted and plated on a plate without selection markers to obtain Bacteroides mutant strains without selection markers and without exogenous plasmids.
4. The gene editing method of claim 1, wherein, The transformed E. coli was S17; Preferably, the process of transforming Escherichia coli includes: mixing the successfully assembled plasmid with Escherichia coli S17 competent cells, placing them on ice in an ice bath, then performing heat shock, cooling them on ice, adding antibiotic-free liquid culture medium, culturing them in an incubator, activating them, and then spreading the bacterial solution onto a plate containing a first selection marker.
5. The gene editing method of claim 1, wherein, The process of conjugation and screening includes: culturing Escherichia coli containing plasmids and wild-type Bacteroides separately to the logarithmic phase, mixing the two bacteria, centrifuging, discarding the supernatant, suspending the bacterial mixture precipitate in liquid culture medium, and then transferring it to a plate without selection markers for aerobic culture; subsequently, scraping the bacterial growth, eluting it in liquid culture medium, taking a portion of the liquid and placing it on a solid plate containing a second selection marker, spreading it evenly, and culturing it under anaerobic conditions.
6. The gene editing method of claim 1, wherein, The gene editing process includes: culturing Bacteroides containing the target plasmid in a liquid medium containing a second selection marker and an inducer until it reaches the logarithmic growth phase, then performing serial dilutions, and then plating it onto solid plates containing the second selection marker and an inducer for anaerobic culture; then picking single clones from the selection plate and culturing them overnight in a medium containing the second selection marker; and finally identifying whether the target gene has been edited.
7. The gene editing method of claim 1, wherein, The first screening marker includes an ampicillin resistance marker; Preferably, the second screening marker includes an erythromycin resistance marker and a gentamicin resistance marker; Preferably, the inducing agent includes an aTc inducing agent.
8. The gene editing method of claim 1, wherein, The process of plasmid loss by passaging includes: adding no-screening marker liquid medium or streaking on no-screening marker plate to the Bacteroides mutant strain which has been edited and contains screening marker resistance, continuously passaging for 10-15 generations, and diluting and plating on no-screening marker plate to obtain the Bacteroides mutant strain without screening marker and exogenous plasmid.
9. The gene editing method of claim 1, wherein, The enterobacterium Bacteroides is selected from Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides ovatus or Bacteroides uniformis.
10. The mutant strain obtained by the gene editing method according to any one of claims 1-9, wherein the mutant strain has deletion and / or insertion of large fragment gene cluster compared with the wild type.