Genome editing method and system for animal bifidobacterium, electronic equipment and computer readable medium

Abstract

The invention provides a genome editing method for animal bifidobacterium. The genome editing method comprises the following steps: constructing plasmids capable of being expressed in bifidobacterium; carrying out demethylation on the plasmid, and carrying out in-vitro methylation modification; the plasmid subjected to methylation modification is electrically transformed into competent cells of animal bifidobacterium; a gene editing mutant strain is obtained through resistance screening and homologous recombination, a'totipotent 'plasmid which can express Cas9 and sgRNA in bifidobacteria and carries a homologous repair template is constructed, the plasmid is protected by simulating a methylation modification system of bifidobacteria in vitro, then the plasmid is electrically transferred into a thallus, and the homologous repair template is obtained. A gene editing mutant strain is obtained through resistance screening and homologous recombination, gene knockout is achieved in animal bifidobacterium, flow gene editing is achieved, methylation modification is conducted on non-methylation plasmids in vitro, and a mutant strain without a selection marker can be obtained. In addition, the invention also provides a genome editing system, electronic equipment and a computer readable medium.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method, system, electronic device, and computer-readable medium for genome editing of Bifidobacterium animalis. Background Technology

[0002] Bifidobacteria are an important component of the gut microbiota in humans and mammals. They can utilize carbohydrates to produce abundant beneficial metabolites, and as a dominant flora in the gut, they are closely related to host health. However, the presence of sensitive restriction modification systems and difficulties in homologous recombination in Bifidobacteria limit gene editing, severely hindering basic research and potential applications of Bifidobacteria.

[0003] The CRISPR / spCas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein spCas9) gene editing system is a CRISPR type II system. The CRISPR / spCas9M-GFP system, where spCas9BM is the name of spCas9 after codon optimization for Bifidobacterium animalis. The principle of the CRISPR / spCas9BM system is as follows: ① Recognition component: The CRISPR sequence is transcribed to form sgRNA with target recognition function; ② Cutting component: spCas9BM is a nuclease that binds to sgRNA. As the sgRNA specifically recognizes and binds to the specific DNA sequence, spCas9BM cuts the target DNA strand, creating a gap in the gene and achieving gene breakage. Subsequently, intracellular repair systems, such as homologous recombination or non-homologous end joining, repair the gene, achieving the purpose of gene editing.

[0004] In 2020, Hiroka Koguchi et al. constructed a recombinase-based in vivo expression technology (R-IVET) and applied the phage P1 Cre / loxP system to Bifidobacterium longum subspecies 105-A to identify its intestinal-specific gene expression in mice (Koguchi et al. 2020). In 2021, Hoedt et al. successfully knocked out the α-galactosidase gene, alcohol dehydrogenase gene, and galactose phosphotransferase gene in Bifidobacterium using a homologous recombination double crossover method (Hoedt et al. 2021). In 2022, Meichen Pan et al. achieved gene knockout of lacA and lacl in Bifidobacterium using the endogenous CRISPR system (Pan et al. 2022).

[0005] Gene knockout methods for Bifidobacterium based on homologous recombination double crossover are time-consuming and labor-intensive (2-5 weeks), and the low homologous recombination efficiency in Bifidobacterium results in very low gene editing efficiency (1%-4%). ​​Cre / loxP technology is based on Cre recombinase and the loxP sequence. Gene editing is achieved by inducing crossover between two DNA molecules through the guidance of the loxP sequence and Cre enzyme cleavage. However, this technology has limitations in insertion sites and cannot achieve scarless editing. The endogenous Crispr system is based on the Crispr-Cas system inherent in the target host. Gene editing is achieved by guiding the host's own Crispr-Cas protein through adjacent continuation sequences in the host's original spacer. However, the endogenous Crispr system lacks universality and requires the target host to possess a usable Crispr system. Summary of the Invention

[0006] In view of this, the present invention provides a method, system, electronic device and computer-readable medium for genome editing of Bifidobacterium animalis, so as to construct an efficient, simple and scarless genome editing system in Bifidobacterium animalis, which can achieve gene knockout.

[0007] To solve the above problems, this application adopts the following technical solution: One of the objectives of this application is to provide a genome editing method for Bifidobacterium animalis, comprising: Construct plasmids that can be expressed in Bifidobacteria; The plasmid was demethylated and then subjected to in vitro methylation modification. The methylated plasmid was electroporated into competent cells of Bifidobacterium animalis; Gene-edited mutant strains were obtained through resistance screening and homologous recombination.

[0008] In some embodiments, the step of constructing a plasmid that can be expressed in Bifidobacteria specifically includes: The Bifidobacterium replicon pAM1-backbone was linked to the inducible promoter PtetW to obtain pAM1-PtetW; The optimized spCas9BM gene was inserted downstream of PtetW to obtain pAM1-PtetW-spCas9BM; The left and right homologous arms of the target gene were inserted into specific positions on the plasmid to obtain pAM1-HR-PtetW-spCas9BM; The constitutive promoter Pkaso*tss17 and gRNA scaffold were assembled into a plasmid to obtain pAM1-PkasO*tss17-gRNA scaffold-HR-PtetW-spCas9BM; By using reverse PCR, a 20bp specific targeting sequence designed for the target gene was inserted into the front end of the gRNA scaffold, and the complete plasmid pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM was finally constructed.

[0009] In some of these embodiments, wherein: The spCas9 gene derived from Streptococcus pyogenes was codon-optimized using an online tool to obtain spCas9BM; Using the CHOPCHOP tool, an sgRNA sequence is designed in a specific region of the target gene to ensure that it efficiently and specifically guides Cas9 for cleavage.

[0010] In some embodiments, the steps of demethylating the plasmid and performing in vitro methylation modification specifically include: By comparing data with databases, genes that may encode methyltransferases were identified in the genome of the target animal's Bifidobacterium strain. The methyltransferase gene was cloned into an E. coli plasmid that is easy to express in vitro, and a ribosome binding site preferred by Bifidobacteria was added to enhance its translation efficiency, resulting in PUC19-RBS-0033; Using a cell-free protein synthesis system, with PUC19-RBS-0033 as a template, an active 0033 methyltransferase protein was synthesized in a test tube to obtain a mixture TXTL-0033. The constructed CRISPR editing plasmid was mixed with TXTL-0033 to obtain the reaction system; The proteins in the reaction system were degraded using proteinase K, and then the methylated CRISPR plasmids were recovered and purified by agarose gel electrophoresis.

[0011] In some embodiments, the step of electroporating the methylated plasmid into competent cells of Bifidobacterium animalis specifically includes: The methylated plasmid was mixed with competent cells and placed in an electroporation cuvette. Under the action of a high-voltage pulse, the cell membrane temporarily formed reversible micropores, allowing the plasmid DNA to enter the cell. Immediately after the electric shock, the cells were added to a rich culture medium and cultured for several hours under suitable temperature and anaerobic conditions to allow the cells to repair membrane damage and begin to express the resistance gene on the plasmid. The revived bacterial culture was spread on MRS plates containing the corresponding antibiotics. Only cells that had been successfully transformed with plasmids could grow and form single colonies.

[0012] In some embodiments, the step of obtaining gene-edited mutant strains through resistance selection and homologous recombination specifically includes: The monoclonal cells were transferred to new resistant plates for streaking culture. During the culture, an inducer was added to initiate Cas9 expression. Cas9 and sgRNA worked together to cause DNA double-strand breaks at the target location in the genome. The cells used homologous arms carried on the plasmid to repair the breaks, thereby introducing the mutation into the genome and verifying it by PCR.

[0013] In some embodiments, the PCR-validated positive clones can be sequenced to confirm that the edited site sequence fully conforms to the design.

[0014] Secondly, this application also provides a genome editing system for Bifidobacterium animalis, comprising: Plasmid building blocks are used to construct plasmids that can be expressed in Bifidobacteria. A modification unit is used to demethylate the plasmid and perform in vitro methylation modification; The electroconversion unit is used to electroconvert methylated plasmids into competent cells of Bifidobacterium animalis. Recombination units are used to obtain gene-edited mutant strains through resistance screening and homologous recombination.

[0015] Thirdly, this application also provides an electronic device, including: One or more processors; Memory, used to store one or more programs; When the one or more programs are executed by the one or more processors, the one or more processors implement any of the methods.

[0016] Fourthly, this application also provides a computer-readable medium storing a computer program, characterized in that the computer program, when executed by a processor, implements the steps in any of the methods described.

[0017] The present application adopts the above technical solution, and its beneficial effects are as follows: This application provides a genome editing method for Bifidobacterium animalis, which involves constructing a plasmid that can be expressed in Bifidobacterium; demethylating the plasmid and performing in vitro methylation modification; electroporating the methylated plasmid into competent cells of Bifidobacterium animalis; and obtaining gene-edited mutant strains through resistance selection and homologous recombination. This application constructs an "all-purpose" plasmid that can express Cas9 and sgRNA in Bifidobacterium and carries a homologous repair template. The plasmid is protected in vitro using a methylation modification system that mimics that of Bifidobacterium, and then electroporated into bacterial cells. Gene-edited mutant strains are obtained through resistance selection and homologous recombination. This method achieves gene knockout in Bifidobacterium animalis, realizes a streamlined gene editing process, and allows for the in vitro methylation modification of unmethylated plasmids to obtain mutant strains without selection markers. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A flowchart illustrating the steps of the genome editing method for Bifidobacterium animalis provided in this application embodiment.

[0020] Figure 2 This is a schematic diagram illustrating the successful establishment of a promoter library in Bifidobacterium animalis AR668, as described in this application embodiment.

[0021] Figure 3 The plasmid structure diagram provided for the embodiments of this application.

[0022] Figure 4 This is a schematic diagram illustrating the process of achieving dam methylation modification of the unmethylated plasmid pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM in vitro, as provided in the embodiments of this application.

[0023] Figure 5 The plasmid PUC19-RBS-OO33 provided in Example 5 of this application is plotted.

[0024] Figure 6 This embodiment is a schematic diagram illustrating the process of knocking out the uracil phosphotransferase gene (UPP) in Bifidobacterium animalis.

[0025] Figure 7 This is a schematic diagram of the genome editing system for Bifidobacterium animalis according to an embodiment of this application.

[0026] Figure 8 This is a schematic diagram of the structure of an electronic device provided in an embodiment of this application. Detailed Implementation

[0027] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified. To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0028] Please see Figure 1 The present application provides a flowchart of a genome editing method for Bifidobacterium animalis, which includes steps S10 to S40. The specific implementation of each step is described in detail below.

[0029] Step S10: Construct a plasmid that can be expressed in Bifidobacteria.

[0030] In this embodiment, a single plasmid system containing all necessary components is assembled that can: induce the expression of the Cas9 protein (gene scissors) in Bifidobacteria; constitutively express sgRNA (guide RNA) targeting the target gene; and provide upstream and downstream homologous arms of the target gene for repairing DNA breaks (repair template). This protocol is highly dependent on the Bifidobacterium animalis strain (AR668) used and its unique restriction-modification system (0033 methyltransferase). Please refer to [link to documentation]. Figure 2 This is a schematic diagram illustrating the successful establishment of a promoter library in Bifidobacterium animalis AR668 in this embodiment.

[0031] In this embodiment, spCas9 was codon optimized to obtain the optimized Cas gene, spCas9BM. Gene elements, including pAM1-backbone, promoter PtetW, codon-optimized spCas9BM, mCherry, Pkaso*tss17, sgRNA, and upstream and downstream homologous arms (1000 bp) of the target gene, were assembled using Gibson. The molar ratio of each element was determined according to the manufacturer's instructions, and assembly was performed at 50°C for 15 minutes.

[0032] Furthermore, Cas9 codon optimization: The spCas9 gene derived from Streptococcus pyogenes was codon optimized using an online tool to better suit the Bifidobacterium translation system, thereby improving expression efficiency and yielding spCas9BM. The specific website is: https: / / www.genewiz.com.cn / public / services / gene-synthesis / mmz.

[0033] Furthermore, gRNA design: Using the CHOPCHOP tool, a 20bp sgRNA sequence was designed in a specific region of the target gene to ensure efficient and specific guidance for Cas9 cleavage. The specific URL is: http: / / chopchop.cbu.uib.no / .

[0034] Furthermore, homologous arm design: On both sides of the target gene editing site, approximately 1000 bp of genomic sequence is selected as homologous arms (L-left arm, R-right arm). This serves as a template for subsequent homologous recombination, and the desired deletions, insertions, or point mutations can be included in the middle.

[0035] Furthermore, the steps in constructing plasmids that can be expressed in Bifidobacteria specifically include: a. Construction of the basic expression vector: The Bifidobacterium replicon pAM1-backbone was linked to the inducible promoter PtetW to obtain pAM1-PtetW. PtetW can initiate the expression of downstream genes upon the addition of tetracycline / hydrotetracycline.

[0036] b. Introducing Cas9: The optimized spCas9BM gene was inserted downstream of PtetW to obtain pAM1-PtetW-spCas9BM. At this point, the plasmid was capable of inducing Cas9 expression.

[0037] c. Introducing a homology repair template: The left homology arm (L-HA) and right homology arm (R-HA) of the target gene are inserted into specific locations on the plasmid to obtain pAM1-HR-PtetW-spCas9BM. The homology arms provide the "blueprint" for subsequent repair.

[0038] d. Introducing the gRNA expression cassette: The constitutive promoter PkasO*tss17 and the gRNA scaffold (the backbone structure of the sgRNA) are assembled into a plasmid to obtain pAM1-PkasO*tss17-gRNA scaffold-HR-PtetW-spCas9BM. This cassette can continuously transcribe the sgRNA backbone.

[0039] e. Insertion of the target sequence: Using reverse PCR, a 20bp specific target sequence designed for the target gene is inserted into the front end of the gRNA scaffold, ultimately completing the construction of the complete plasmid pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM. At this point, the plasmid can simultaneously express the sgRNA targeting the target gene and the inducible Cas9 expression.

[0040] Please see Figure 3 , is the plasmid structure diagram constructed in this embodiment.

[0041] In this embodiment, the plasmid mainly consists of six parts: the pAM1-backbone plasmid backbone, the tetracycline-inducible promoter PtetW, the spCas9 protein gene, gRNA, and homology arms (L / R). The Gibson assembly method is primarily used to assemble this series of elements into a single plasmid. Specific information about each element is as follows: 1) Design primers to first linearize the pAM1-backbone plasmid backbone and PtetW. After linearization, there will be a 20bp repetitive sequence between the two elements (this needs to be considered before designing primers). Then, assemble the components in a 10 μL system, which contains 0.1 pmol of the pAM1-backbone plasmid backbone, 0.2 pmol of the PtetW induction system element, and 5 μL of 2 × CE Mix V3. Then, place the system in a PCR instrument and react at 50℃ for 15 min. After that, transform the assembled product into DH5α competent cells and screen them on LB ampicillin (AMP) resistant plates to obtain the plasmid pAM1-PtetW.

[0042] 2) Linearize the obtained plasmids pAM1-PtetW and spCas9BM elements, and similarly make a 20bp repeat sequence between the two fragments. Assemble them according to the above method, with a molar ratio of 1:1 between the two elements. Other methods are the same as above, and the pAM1-PtetW-spCas9BM plasmid can be obtained.

[0043] 3) Assemble the plasmid pAM1-PtetW-spCas9BM with the upstream and downstream homologous arms of the target gene. After linearizing pAM1-PtetW-spCas9BM and the upstream and downstream homologous arms, ensure that there is a 20bp repeat sequence between the three fragments. Assemble using the method described above, with the molar ratio of each element being 1:1:1. Other methods are the same as above to obtain the pAM1-HR-PtetW-spCas9BM plasmid.

[0044] 4) Assemble the plasmid pAM1-HR-PtetW-spCas9BM, gRNA scaffold, and PkasO*tss17 (since PkasO*tss17 is relatively short, it is directly designed onto the primers). After linearization, the plasmid pAM1-HR-PtetW-spCas9BM, gRNA scaffold, and PkasO*tss17 have 20bp repeat sequences between each element. Assemble them using the method described above, with a molar ratio of 1:1 for each element and a total molar count not exceeding 0.2 pmol. Other methods are the same as above to obtain the plasmid pAM1-PkasO*tss17-gRNA scaffold-HR-PtetW-spCas9BM.

[0045] 5) Assemble pAM1-PkasO*tss17-gRNA scaffold-HR-PtetW-spCas9BM and sgRNA. Design sgRNA on primers. There is a 20bp repeat sequence between each element. Perform reverse PCR. Use the above method to assemble. The system is 10 μL, 5 μL of PCR product, and 5 μL of 2 × CE Mix V3. Then place it in a PCR instrument and react at 50℃ for 15 min to obtain pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM.

[0046] It is understandable that, through the above steps, the cutting tool (Cas9), navigation system (sgRNA), and repair blueprint (homologous arm) are integrated into a vector that can replicate and be expressed in the target bacterium (Bifidobacterium).

[0047] Step S20: Demethylate the plasmid and perform in vitro methylation modification.

[0048] It is understandable that Bifidobacteria possess an active restriction-modification system capable of recognizing and cleaving exogenous unmethylated DNA. This step involves in vitro methylation, which imbues the plasmid with Bifidobacteria's own methylation markers, thereby preventing it from being degraded as an "invader."

[0049] In this embodiment, the step of demethylating the plasmid and performing in vitro methylation modification specifically includes the following steps: Predicting methyltransferases: By comparing data with databases, identify genes (such as gene 0033) that may encode methyltransferases in the genome of the target animal Bifidobacterium strain (AR668).

[0050] Constructing a methyltransferase expression plasmid: The methyltransferase gene (0033) was cloned into an E. coli plasmid (such as PUC19) that is easy to express in vitro, and a ribosome binding site (RBS) preferred by Bifidobacteria was added to enhance its translation efficiency, resulting in PUC19-RBS-0033.

[0051] Methyltransferase was produced by in vitro transcription and translation: using a cell-free protein synthesis system (PURE Express kit) with PUC19-RBS-0033 as a template, active 0033 methyltransferase protein was synthesized in a test tube to obtain a mixture TXTL-0033.

[0052] In vitro methylation reaction: The CRISPR editing plasmid constructed in the first step was mixed with TXTL-0033. In the reaction system, the 0033 methyltransferase recognizes a specific sequence on the plasmid and methylates it, mimicking its native state in Bifidobacteria.

[0053] Purification of methylated plasmids: Proteins in the reaction system were degraded with proteinase K, and then the methylated CRISPR plasmids were purified by agarose gel electrophoresis.

[0054] Please see Figure 4 This is a schematic diagram illustrating the process of achieving dam methylation modification of the unmethylated plasmid pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM in vitro, specifically including the following steps: 1) The methyltransferase encoding gene in AR688 was predicted using the REBASE database (https: / / rebase.neb.com / rebase / rebase.html) and the whole genome sequencing results of Bifidobacterium animalis AR668.

[0055] 2) Design primers to linearize the PUC19-backbone and AR668 encoding gene 0033. After linearization, there will be a 20bp repetitive sequence between the two elements. Use the above method to assemble and screen on LB ampicillin (AMP) resistance plates to obtain plasmid PUC19-OO33.

[0056] 3) Primer design: Ribosome binding sites suitable for Bifidobacteria were inserted into PUC19-OO33 to promote the expression of 0033. Based on the RBS sequence suitable for Bifidobacteria reported by Justin M. Vento et al. (Vento et al. 2024), primers were designed. Due to the short RBS sequence, the RBS was directly designed into the primers. Using PUC19-0033 as a template, PUC19-0033 was linearized and assembled using the above method. The plasmid PUC19-RBS-OO33 was obtained by screening on LB ampicillin (AMP) resistance plates. Please refer to [link to relevant documentation]. Figure 5 .

[0057] 4) Mix the PURE Express kit and PUC19-RBS-OO33 according to the ratio shown in Table 1, and react at 29℃ for 12-16 h to establish an in vitro transcription and translation system, which is named TXTL-0033.

[0058] 5) After reacting for 12-16 h, TXTL-0033 and pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM were gently mixed according to the ratio shown in Table 2. The mixture was reacted at 37℃ for 1-3 h. Then, 150 μL of proteinase K working solution (1 μL proteinase K + 149 μL redistilled water) was added, and the mixture was gently mixed. The mixture was then reacted at 50℃ for 30 min to obtain a mixed solution of protein-degraded TXTL-0033 and pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM.

[0059] 6) The pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM was purified using the agarose gel extraction method to obtain the methylated plasmid pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM-0033.

[0060] In this embodiment, the successfully constructed plasmid was demethylated using *E. coli* JM110, followed by in vitro methylation modification using the PURE Express kit (details of the PURE Express reaction system are shown in Table 1). All components were mixed in proportion and incubated at 29°C for 12-16 hours (named TXTL) to construct the in vitro transcription-translation system. Subsequently, the components were gently mixed according to the in vitro methylation transcription reaction system (Table 2) and incubated at 37°C for 1-3 hours. The purified in vitro methylated plasmid was then recovered via agarose gel electrophoresis.

[0061] Table 1. PUREExpress Reaction System

[0062] Table 2 In vitro methylation transcription reaction system

[0063] It is understandable that unmodified exogenous plasmids would be rapidly destroyed after entering Bifidobacteria. By pre-methylating them in vitro, the plasmids can be "disguised" as the host's own DNA, greatly improving the survival rate after electroporation.

[0064] Step S30: Electroporate the methylated plasmid into competent cells of Bifidobacterium animalis.

[0065] In this embodiment, the methylated plasmid is electroporated into competent cells of Bifidobacterium animalis, specifically including the following steps: Preparation of competent cells: This is not described in detail in the text, but usually involves using reagents such as glycine to weaken the cell wall and preparing electrocompetent cells through multiple washings, which are then stored in hypertonic buffer.

[0066] Electroporation: The purified methylated plasmid was mixed with competent cells and placed in an electroporation cuvette. Under the action of a high-voltage electric pulse (2.5 kV), reversible micropores were temporarily formed in the cell membrane, allowing the plasmid DNA to enter the cell.

[0067] Resuscitation: Immediately after electric shock, add rich medium (MRS) and culture for several hours under suitable temperature (37℃) and anaerobic conditions to allow cells to repair membrane damage and begin expressing resistance genes on plasmids (such as the erythromycin resistance gene EM).

[0068] Spreading screening: The revived bacterial culture was spread onto MRS plates containing the appropriate antibiotic (erythromycin). Only cells that had been successfully transformed with the plasmid could grow and form single colonies.

[0069] In this embodiment, 1000 ng of successfully methylated plasmid was added to 100 μL of competent Bifidobacterium animalis cells. After gentle mixing, the mixture was transferred to a 2 mm electroporation cuvette and incubated on ice for 10 min. The BIORAD program was set to 2.5 kV for 4 ms for electroporation, and 900 μL of MRS liquid medium was quickly added. The cells were then anaerobically incubated at 37°C for 3 h. After centrifugation at 6000 rpm for 3 min, 900 μL of supernatant was discarded, and the bacterial pellet was resuspended in the remaining 100 μL of liquid. The resuspended pellet was then plated onto an MRS antibody plate (EM).

[0070] Understandably, this step uses physical methods to forcibly deliver the editing toolkit (plasmid) into the target bacteria.

[0071] Step S40: Obtain gene-edited mutant strains through resistance screening and homologous recombination.

[0072] In this embodiment, the steps of obtaining gene-edited mutant strains through resistance screening and homologous recombination specifically include: transferring monoclonal cells to new resistant plates for scrambling culture; adding an inducer to initiate Cas9 expression during culture; Cas9 and sgRNA working together to cause DNA double-strand breaks at the target location in the genome; the cells using homologous arms carried on the plasmid to repair the breaks, thereby introducing the mutation into the genome and performing PCR verification.

[0073] It's understandable that single clones growing on erythromycin-containing plates only prove they contain plasmids, but not necessarily that gene editing has occurred. The single clones are then transferred to new antibiotic-resistant plates for streaking culture. During culture, an inducer (such as tetracycline) can be added to initiate Cas9 expression. Cas9 and sgRNA work together to create a double-strand break in DNA at the target genomic location. Cells use homologous arms carried on the plasmid to repair the break, thus introducing the mutation into the genome. Genomic DNA is extracted from the streaked bacterial culture, and a pair of verification primers on the outer side of the homologous arms is designed. PCR amplification is then performed. If correct homologous recombination occurs, the size of the PCR product will differ from the wild type (e.g., shorter due to a missing sequence or longer due to a marker insertion). Analysis of the PCR product size by agarose gel electrophoresis provides a preliminary identification of the mutant. Clones that test positive for PCR can be sequenced to confirm that the edited site sequence fully conforms to the design.

[0074] It is understandable that the plasmid is maintained through resistance, and the designed mutation is integrated into the genome using the bacteria's own repair mechanism. Finally, the editing success is confirmed through molecular biology methods.

[0075] Furthermore, it includes sequencing PCR-positive clones to confirm that the edited site sequence fully conforms to the design. Please refer to [link to documentation]. Figure 6 This is a schematic diagram illustrating the process of knocking out the uracil phosphotransferase gene (UPP) in Bifidobacterium animalis in this embodiment.

[0076] This step is understandable; it's a result verification process. The plasmid is maintained through resistance, and the designed mutation is integrated into the genome using the bacteria's own repair mechanisms. Finally, molecular biology methods are used to confirm successful editing.

[0077] This application provides a genome editing method for Bifidobacterium animalis, which involves constructing a plasmid that can be expressed in Bifidobacterium; demethylating the plasmid and performing in vitro methylation modification; electroporating the methylated plasmid into competent cells of Bifidobacterium animalis; and obtaining gene-edited mutant strains through resistance selection and homologous recombination. This application constructs an "all-purpose" plasmid that can express Cas9 and sgRNA in Bifidobacterium and carries a homologous repair template. The plasmid is protected in vitro using a methylation modification system that mimics that of Bifidobacterium, and then electroporated into bacterial cells. Gene-edited mutant strains are obtained through resistance selection and homologous recombination. This method achieves gene knockout in Bifidobacterium animalis, realizes a streamlined gene editing process, and allows for the in vitro methylation modification of unmethylated plasmids to obtain mutant strains without selection markers.

[0078] Please see Figure 7 This application also provides a genome editing system for Bifidobacterium animalis, comprising: a plasmid construction unit, a modification unit, an electroporation unit, and a recombination unit. The specific implementation of each unit is described in detail below.

[0079] The plasmid building block is used to construct plasmids that can be expressed in Bifidobacteria.

[0080] Furthermore, the steps in constructing plasmids that can be expressed in Bifidobacteria specifically include: a. Construction of the basic expression vector: The Bifidobacterium replicon pAM1-backbone was linked to the inducible promoter PtetW to obtain pAM1-PtetW. PtetW can initiate the expression of downstream genes upon the addition of tetracycline / hydrotetracycline.

[0081] b. Introducing Cas9: The optimized spCas9BM gene was inserted downstream of PtetW to obtain pAM1-PtetW-spCas9BM. At this point, the plasmid was capable of inducing Cas9 expression.

[0082] c. Introducing a homology repair template: The left homology arm (L-HA) and right homology arm (R-HA) of the target gene are inserted into specific locations on the plasmid to obtain pAM1-HR-PtetW-spCas9BM. The homology arms provide the "blueprint" for subsequent repair.

[0083] d. Introducing the gRNA expression cassette: The constitutive promoter PkasO*tss17 and the gRNA scaffold (the backbone structure of the sgRNA) are assembled into a plasmid to obtain pAM1-PkasO*tss17-gRNA scaffold-HR-PtetW-spCas9BM. This cassette can continuously transcribe the sgRNA backbone.

[0084] e. Insertion of the target sequence: Using reverse PCR, a 20bp specific target sequence designed for the target gene is inserted into the front end of the gRNA scaffold, ultimately completing the construction of the complete plasmid pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM. At this point, the plasmid can simultaneously express the sgRNA targeting the target gene and the inducible Cas9 expression.

[0085] It is understandable that, through the above steps, the cutting tool (Cas9), navigation system (sgRNA), and repair blueprint (homologous arm) are integrated into a vector that can replicate and be expressed in the target bacterium (Bifidobacterium).

[0086] The modification unit is used to demethylate the plasmid and perform in vitro methylation modification.

[0087] In this embodiment, the step of demethylating the plasmid and performing in vitro methylation modification specifically includes the following steps: Predicting methyltransferases: By comparing data with databases, identify genes (such as gene 0033) that may encode methyltransferases in the genome of the target animal Bifidobacterium strain (AR668).

[0088] Constructing a methyltransferase expression plasmid: The methyltransferase gene (0033) was cloned into an E. coli plasmid (such as PUC19) that is easy to express in vitro, and a ribosome binding site (RBS) preferred by Bifidobacteria was added to enhance its translation efficiency, resulting in PUC19-RBS-0033.

[0089] Methyltransferase was produced by in vitro transcription and translation: using a cell-free protein synthesis system (PURE Express kit) with PUC19-RBS-0033 as a template, active 0033 methyltransferase protein was synthesized in a test tube to obtain a mixture TXTL-0033.

[0090] In vitro methylation reaction: The CRISPR editing plasmid constructed in the first step was mixed with TXTL-0033. In the reaction system, the 0033 methyltransferase recognizes a specific sequence on the plasmid and methylates it, mimicking its native state in Bifidobacteria.

[0091] Purification of methylated plasmids: Proteins in the reaction system were degraded with proteinase K, and then the methylated CRISPR plasmids were purified by agarose gel electrophoresis.

[0092] It is understandable that unmodified exogenous plasmids would be rapidly destroyed after entering Bifidobacteria. By pre-methylating them in vitro, the plasmids can be "disguised" as the host's own DNA, greatly improving the survival rate after electroporation.

[0093] The electroconversion unit is used to electroconvert methylated plasmids into competent cells of Bifidobacterium animalis.

[0094] In this embodiment, the methylated plasmid is electroporated into competent cells of Bifidobacterium animalis, specifically including the following steps: Preparation of competent cells: This is not described in detail in the text, but usually involves using reagents such as glycine to weaken the cell wall and preparing electrocompetent cells through multiple washings, which are then stored in hypertonic buffer.

[0095] Electroporation: The purified methylated plasmid was mixed with competent cells and placed in an electroporation cuvette. Under the action of a high-voltage electric pulse (2.5 kV), reversible micropores were temporarily formed in the cell membrane, allowing the plasmid DNA to enter the cell.

[0096] Resuscitation: Immediately after electric shock, add rich medium (MRS) and culture for several hours under suitable temperature (37℃) and anaerobic conditions to allow cells to repair membrane damage and begin expressing resistance genes on plasmids (such as the erythromycin resistance gene EM).

[0097] Spreading screening: The revived bacterial culture was spread onto MRS plates containing the appropriate antibiotic (erythromycin). Only cells that had been successfully transformed with the plasmid could grow and form single colonies.

[0098] Understandably, this step uses physical methods to forcibly deliver the editing toolkit (plasmid) into the target bacteria.

[0099] Recombination units are used to obtain gene-edited mutant strains through resistance screening and homologous recombination.

[0100] In this embodiment, the steps of obtaining gene-edited mutant strains through resistance screening and homologous recombination specifically include: transferring monoclonal cells to new resistant plates for scrambling culture; adding an inducer to initiate Cas9 expression during culture; Cas9 and sgRNA working together to cause DNA double-strand breaks at the target location in the genome; the cells using homologous arms carried on the plasmid to repair the breaks, thereby introducing the mutation into the genome and performing PCR verification.

[0101] It is understandable that the plasmid is maintained through resistance, and the designed mutation is integrated into the genome using the bacteria's own repair mechanism. Finally, the editing success is confirmed through molecular biology methods.

[0102] Furthermore, this includes sequencing PCR-positive clones to confirm that the edited site sequence fully conforms to the design. This step can be understood as result acceptance. The plasmid is maintained using antibiotics, and the designed mutation is integrated into the genome using the bacteria's own repair mechanisms. Finally, molecular biology methods are used to confirm successful editing.

[0103] This application provides a genome editing system for Bifidobacterium animalis, which involves constructing a plasmid that can be expressed in Bifidobacterium; demethylating the plasmid and performing in vitro methylation modification; electroporating the methylated plasmid into competent cells of Bifidobacterium animalis; and obtaining gene-edited mutant strains through resistance selection and homologous recombination. This application constructs an "all-purpose" plasmid that can express Cas9 and sgRNA in Bifidobacterium and carries a homologous repair template. The plasmid is protected using an in vitro methylation modification system that mimics that of Bifidobacterium animalis, and then electroporated into bacterial cells. Gene-edited mutant strains are obtained through resistance selection and homologous recombination. Gene knockout is achieved in Bifidobacterium animalis, realizing a streamlined gene editing process. Furthermore, in vitro methylation modification of the unmethylated plasmid can yield mutant strains without selection markers.

[0104] Based on the same inventive concept, embodiments of the present invention also provide an electronic device. Figure 8 This is a structural block diagram of an electronic device provided in an embodiment of the present invention. Figure 8 As shown, an embodiment of the present invention provides an electronic device including: one or more processors 101, a memory 102, and one or more I / O interfaces 103. The memory 102 stores one or more programs, which, when executed by the one or more processors, cause the one or more processors to implement any of the genome editing methods for Bifidobacterium animalis as described in the above embodiments; the one or more I / O interfaces 103 are connected between the processor and the memory, configured to enable information interaction between the processor and the memory.

[0105] The processor 101 is a device with data processing capabilities, including but not limited to a central processing unit (CPU); the memory 102 is a device with data storage capabilities, including but not limited to random access memory (RAM, more specifically SDRAM, DDR, etc.), read-only memory (ROM), electrically erasable programmable read-only memory (EEPROM), and flash memory (FLASH); the I / O interface (read / write interface) 103 is connected between the processor 101 and the memory 102, and can realize information interaction between the processor 101 and the memory 102, including but not limited to a data bus (Bus).

[0106] In some embodiments, the processor 101, memory 102, and I / O interface 103 are interconnected via bus 104, and thus connected to other components of the computing device.

[0107] In some embodiments, the one or more processors 101 include a field-programmable gate array.

[0108] This invention also provides a computer-readable medium. The computer-readable medium stores a computer program, which, when executed by a processor, implements the steps of any of the genome editing methods for Bifidobacterium animalis described in the above embodiments. The computer-readable storage medium may be volatile or non-volatile.

[0109] This invention also provides a computer program product, including computer-readable code, or a non-volatile computer-readable storage medium carrying computer-readable code. When the computer-readable code is run in the processor of an electronic device, the processor in the electronic device executes the above-described genome editing method for Bifidobacterium animalis.

[0110] Those skilled in the art will understand that all or some of the steps, systems, and apparatuses disclosed above, and their functional modules / units, can be implemented as software, firmware, hardware, or suitable combinations thereof. In hardware implementations, the division between functional modules / units mentioned above does not necessarily correspond to the division of physical components; for example, a physical component may have multiple functions, or a function or step may be performed collaboratively by several physical components. Some or all physical components may be implemented as software executed by a processor, such as a central processing unit, digital signal processor, or microprocessor, or as hardware, or as an integrated circuit, such as an application-specific integrated circuit (ASIC). Such software can be distributed on a computer-readable storage medium, which may include computer storage media (or non-transitory media) and communication media (or transient media).

[0111] As is well known to those skilled in the art, the term computer storage medium includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing information such as computer-readable program instructions, data structures, program modules or other data. Computer storage media include, but are not limited to, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM), static random access memory (SRAM), flash memory or other memory technologies, and portable compact disc read-only memory (CD). ROM, digital multifunction disc (DVD) or other optical disc storage, magnetic cartridges, magnetic tapes, disk storage or other magnetic storage devices, or any other medium that can be used to store desired information and can be accessed by a computer. Furthermore, it is well known to those skilled in the art that communication media typically contain computer-readable program instructions, data structures, program modules, or other data in modulated data signals such as carrier waves or other transmission mechanisms, and may include any information delivery medium.

[0112] The computer-readable program instructions described herein can be downloaded from computer-readable storage media to various computing / processing devices, or downloaded via a network, such as the Internet, local area network, wide area network, and / or wireless network, to an external computer or external storage device. The network may include copper transmission cables, fiber optic transmission, wireless transmission, routers, firewalls, switches, gateway computers, and / or edge servers. A network adapter card or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards them to the computer-readable storage media in the respective computing / processing device.

[0113] The computer program instructions used to perform the operations of this invention may be assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, state setting data, or source code or object code written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Smalltalk, C++, etc., and conventional procedural programming languages ​​such as the "C" language or similar programming languages. The computer-readable program instructions may be executed entirely on the user's computer, partially on the user's computer, as a standalone software package, partially on the user's computer and partially on a remote computer, or entirely on a remote computer or server. In cases involving a remote computer, the remote computer may be connected to the user's computer via any type of network—including a local area network (LAN) or a wide area network (WAN)—or may be connected to an external computer (e.g., via the Internet using an Internet service provider). In some embodiments, electronic circuitry, such as programmable logic circuitry, field-programmable gate arrays (FPGAs), or programmable logic arrays (PLAs), is personalized by utilizing state information from the computer-readable program instructions. This electronic circuitry can execute the computer-readable program instructions to implement various aspects of the invention.

[0114] The computer program product described herein can be implemented specifically through hardware, software, or a combination thereof. In one alternative embodiment, the computer program product is specifically embodied in a computer storage medium; in another alternative embodiment, the computer program product is specifically embodied in a software product, such as a software development kit (SDK), etc.

[0115] Various aspects of the present invention are described herein with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It should be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer-readable program instructions.

[0116] These computer-readable program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing apparatus to produce a machine such that, when executed by the processor of the computer or other programmable data processing apparatus, they create means for implementing the functions / actions specified in one or more blocks of the flowchart and / or block diagram. These computer-readable program instructions can also be stored in a computer-readable storage medium that causes a computer, programmable data processing apparatus, and / or other device to operate in a particular manner; thus, the computer-readable medium storing the instructions comprises an article of manufacture that includes instructions for implementing aspects of the functions / actions specified in one or more blocks of the flowchart and / or block diagram.

[0117] Computer-readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other device to cause a series of operational steps to be performed on the computer, other programmable data processing apparatus, or other device to produce a computer-implemented process, thereby causing the instructions executed on the computer, other programmable data processing apparatus, or other device to perform the functions / actions specified in one or more boxes of a flowchart and / or block diagram.

[0118] The flowcharts and block diagrams in the accompanying drawings illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the present invention. In this regard, each block in a flowchart or block diagram may represent a module, segment, or portion of an instruction, which contains one or more executable instructions for implementing a specified logical function. In some alternative implementations, the functions marked in the blocks may occur in a different order than those shown in the drawings. For example, two consecutive blocks may actually be executed substantially in parallel, and they may sometimes be executed in reverse order, depending on the functions involved. It should also be noted that each block in the block diagrams and / or flowcharts, and combinations of blocks in the block diagrams and / or flowcharts, may be implemented using a dedicated hardware-based system that performs the specified function or action, or using a combination of dedicated hardware and computer instructions.

[0119] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A genome editing method for Bifidobacterium animalis, characterized in that, include: Construct plasmids that can be expressed in Bifidobacteria; The plasmid was demethylated and then subjected to in vitro methylation modification. The methylated plasmid was electroporated into competent cells of Bifidobacterium animalis; Gene-edited mutant strains were obtained through resistance screening and homologous recombination.

2. The genome editing method for Bifidobacterium animalis as described in claim 1, characterized in that, The steps involved in constructing plasmids that can be expressed in Bifidobacteria specifically include: The Bifidobacterium replicon pAM1-backbone was linked to the inducible promoter PtetW to obtain pAM1-PtetW; The optimized spCas9BM gene was inserted downstream of PtetW to obtain pAM1-PtetW-spCas9BM; The left and right homologous arms of the target gene were inserted into specific positions on the plasmid to obtain pAM1-HR-PtetW-spCas9BM; The constitutive promoter Pkaso*tss17 and gRNA scaffold were assembled into a plasmid to obtain pAM1-PkasO*tss17-gRNA scaffold-HR-PtetW-spCas9BM; By using reverse PCR, a 20bp specific targeting sequence designed for the target gene was inserted into the front end of the gRNA scaffold, and the complete plasmid pAM1-PkasO*tss17-sgRNA-gRNA scaffold-HR-PtetW-spCas9BM was finally constructed.

3. The genome editing method for Bifidobacterium animalis as described in claim 2, characterized in that, in: The spCas9 gene derived from Streptococcus pyogenes was codon-optimized using an online tool to obtain spCas9BM; Using the CHOPCHOP tool, an sgRNA sequence is designed in a specific region of the target gene to ensure that it efficiently and specifically guides Cas9 for cleavage.

4. The genome editing method for Bifidobacterium animalis as described in claim 3, characterized in that, The steps of demethylating the plasmid and performing in vitro methylation modification specifically include: By comparing data with databases, genes that may encode methyltransferases were identified in the genome of the target animal's Bifidobacterium strain. The methyltransferase gene was cloned into an E. coli plasmid that is easy to express in vitro, and a ribosome binding site preferred by Bifidobacteria was added to enhance its translation efficiency, resulting in PUC19-RBS-0033; Using a cell-free protein synthesis system, with PUC19-RBS-0033 as a template, an active 0033 methyltransferase protein was synthesized in a test tube to obtain a mixture TXTL-0033. The constructed CRISPR editing plasmid was mixed with TXTL-0033 to obtain the reaction system; The proteins in the reaction system were degraded using proteinase K, and then the methylated CRISPR plasmids were recovered and purified by agarose gel electrophoresis.

5. The genome editing method for Bifidobacterium animalis as described in claim 4, characterized in that, The step of electroporating the methylated plasmid into competent cells of Bifidobacterium animalis specifically includes: The methylated plasmid was mixed with competent cells and placed in an electroporation cuvette. Under the action of a high-voltage pulse, the cell membrane temporarily formed reversible micropores, allowing the plasmid DNA to enter the cell. Immediately after the electric shock, the cells were added to a rich culture medium and cultured for several hours under suitable temperature and anaerobic conditions to allow the cells to repair membrane damage and begin to express the resistance gene on the plasmid. The revived bacterial culture was spread on MRS plates containing the corresponding antibiotics. Only cells that had been successfully transformed with plasmids could grow and form single colonies.

6. The genome editing method for Bifidobacterium animalis as described in claim 5, characterized in that, The steps for obtaining gene-edited mutant strains through resistance selection and homologous recombination specifically include: The monoclonal cells were transferred to new resistant plates for streaking culture. During the culture, an inducer was added to initiate Cas9 expression. Cas9 and sgRNA worked together to cause DNA double-strand breaks at the target location in the genome. The cells used homologous arms carried on the plasmid to repair the breaks, thereby introducing the mutation into the genome and verifying it by PCR.

7. The genome editing method for Bifidobacterium animalis as described in claim 6, characterized in that, It also includes sequencing of PCR-positive clones to confirm that the edited site sequence fully conforms to the design.

8. A genome editing system for Bifidobacterium animalis, characterized in that, include: Plasmid building blocks are used to construct plasmids that can be expressed in Bifidobacteria. A modification unit is used to demethylate the plasmid and perform in vitro methylation modification; The electroconversion unit is used to electroconvert methylated plasmids into competent cells of Bifidobacterium animalis. Recombination units are used to obtain gene-edited mutant strains through resistance screening and homologous recombination.

9. An electronic device, characterized in that, include: One or more processors; Memory, used to store one or more programs; When the one or more programs are executed by the one or more processors, the one or more processors implement the method as described in any one of claims 1 to 7.

10. A computer-readable medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the steps of the method as described in any one of claims 1 to 7.

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