CN107400677B - Bacillus licheniformis genome editing vector based on CRISPR-Cas9 system and preparation method thereof - Google Patents
Bacillus licheniformis genome editing vector based on CRISPR-Cas9 system and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a Bacillus licheniformis genome editing vector based on a CRISPR-Cas9 system, which is prepared by the following steps: integrating a Cas9 protein expression frame and a sgRNA scaffold expression frame in a starting vector pHY xyl to obtain pHY-Cas9-sgRNA scaffold, and then integrating any one of a plurality of sgRNA fragments which are designed and synthesized by taking amyL as a target site into the pHY-Cas9-sgRNA scaffold to prepare the bacillus licheniformis genome editing vector. The method can greatly improve the current situation that the current bacillus licheniformis gene is difficult to edit, and obviously improve the gene editing efficiency.
Description
Technical Field
The invention relates to the technical field of genes, in particular to a bacillus licheniformis genome editing vector which is connected with a specific sgRNA fragment and is based on a CRISPR-Cas9 system.
Background
Bacillus licheniformis is a gram-positive bacillus, which has only a single-layer cell membrane, can generate endospore under extremely severe conditions of dryness, lack of nutrition and the like, and can resume growth again when the conditions are suitable. Bacillus licheniformis has a wide range of industrial microorganisms. It is not only a major producer of bulk industrial enzyme preparations-amylases and proteases; meanwhile, the enzyme preparation is used for the expression host of foreign genes to produce various food enzyme preparations due to the characteristics of excellent secretion capacity, simple fermentation conditions and food safety. With the complete analysis of the genome of the model strain ATCC14580, people's knowledge on the growth and metabolism of the strain is improved to a new level, and further, a new application is developed for the industrial microorganism.
Currently, with genome sequence information as a blueprint, metabolic engineering based on artificially designed genetic perturbation has become the most effective strategy for studying complex metabolic pathways of bacillus licheniformis and constructing phenotypes for strain transformation. Most of the genetic engineering tools developed for editing bacterial genomes have proven effective for the genetic manipulation of B.licheniformis, but there are limitations that have affected the efficiency with which these tools can be used in this particular host, B.licheniformis. For example, in addition to the traditional gene editing method using PCR fragment (mutation cassette) -mediated homologous integration double exchange, a gene knockout method based on allelic replacement (allelic exchange) is also provided, and the method is characterized in that the knockout and integration of a target gene are completed with the help of an inverse selection marker-mazF. The induced expression of mazF gene in colibacillus can kill cell and make screening, and its sensitivity and accuracy are greatly raised. However, this system still relies on antibiotic markers to enable selection of integrated mutation cassettes during manipulation; although theoretically this antibiotic marker could be deleted in subsequent experiments using FLP recombinase, a significant "scar" would remain on the genome. In addition, the function of FLP recombinase is significantly different among different hosts, greatly limiting the versatility of this gene editing method.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-mediated gene excision systems (Cas) are a class of immune systems that are ubiquitous in bacteria and archaea and that efficiently recognize and cleave foreign DNA, such as phages or plasmids, that enter cells. Its mode of action is as follows: short DNA sequences located between CRISPR palindromic repeats constitute the CRISPR array of genes of interest (CRISPR array), which is transcribed and processed between the repeats to form CRISPR-rna (crrna). The crRNA then guides the Cas nuclease to the target site, and the target sequence is cut with the help of a specific PAM (Protospace-adjacent Motif) sequence. The native CRISPR/Cas9 system from Streptococcus pyogenes (Streptococcus pyogenes) comprises a double RNA complex composed of crRNA and trans-regulatory activating crRNA (tracrrna) that mediates the action of Cas9 endonuclease, one of the earliest CRISPR/Cas9 systems studied. Recently, the system has been successfully applied to gene editing of various microorganisms, including gram-negative escherichia coli and gram-positive s.pneumonia and lactobacillus reuteri, and the results all show that the system can realize rapid and convenient directional modification of bacterial genome.
However, the current bacillus licheniformis genome editing system based on the CRISPR-Cas9 system is not constructed, so that the advanced technology cannot be exerted in the important industrial microorganism of bacillus licheniformis.
Disclosure of Invention
In order to solve the problems in the prior art, the applicant of the present invention provides a bacillus licheniformis genome editing vector based on a CRISPR-Cas9 system and a preparation method thereof. The method can greatly improve the current situation that the current bacillus licheniformis gene is difficult to edit, and obviously improve the gene editing efficiency.
The technical scheme of the invention is as follows:
a Bacillus licheniformis genome editing vector based on a CRISPR-Cas9 system is prepared by the following steps: integrating a Cas9 protein expression frame and a sgRNA scaffold expression frame in a starting vector pHY xyl to obtain pHY-Cas9-sgRNA scaffold, and then integrating any one of a plurality of sgRNA fragments which are designed and synthesized by taking amyL as a target site into the pHY-Cas9-sgRNA scaffold to prepare the bacillus licheniformis genome editing vector.
The Cas9 protein expression cassette includes PxylThe promoter, Cas9 protein, and the amyL terminator.
The sequence of the Cas9 protein expression cassette is shown as SEQ ID NO. 1.
The gRNA scaffold expression cassette includes PHpaIIPromoter and sgRNA scaffold.
The sequence of the gRNA scaffold expression frame is shown as SEQ ID NO. 2.
The sequence of the sgRNA fragment is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
The sequence of the starting vector pHY-xyl is shown in SEQ ID NO. 9.
A preparation method of a Bacillus licheniformis genome editing vector based on a CRISPR-Cas9 system, comprising the following steps:
(1) designing and synthesizing a Cas9 protein expression frame, a sgRNA scaffold expression frame and a plurality of sgRNA fragments which accord with the codon characteristics of the bacillus licheniformis;
optimizing a coding gene of Cas9 protein according to codon preference of Bacillus licheniformis, carrying out in-vitro synthesis, and synthesizing a sgRNA scaffold expression frame and a plurality of sgRNA fragments according to a proper target inside a target gene amyL expression frame;
(2) cloning the Cas9 protein expression frame into an enzyme-cut starting vector pHY xyl to obtain pHY-Cas 9;
cloning the Cas9 protein encoding gene with optimized codon preference in the step (1) into a starting vector pHY-xyl to obtain pHY-Cas9, and expressing the Cas9 protein encoding gene under the mediation of a xylose-induced promoter;
(3) cloning the sgRNA scaffold expression frame into the pHY-Cas9 prepared in the step (2) to obtain pHY-Cas9-sgRNA scaffold;
taking a pMA5 plasmid as a template, using a primer for amplification to obtain a promoter, inserting the amplified product into a plasmid pHY-Cas9 after enzyme digestion, connecting the sgRNA scaffold expression frame synthesized in the step (1) into a corresponding site of the plasmid after enzyme digestion to obtain pHY-Cas9-sgRNA scaffold;
(4) cloning any one of a plurality of sgRNA fragments designed and synthesized by taking amyL as a target site into pHY-Cas9-sgRNA scaffold, and then connecting a gene editing and repairing sequence to obtain the bacillus licheniformis genome editing vector;
mutextracting Bacillus licheniformis genome DNA, amplifying by using DNA polymerase and primers to obtain a target gene part open reading frame truncamyL L, cloning T-A after an amplification product is purified to a pMD18-T-simple vector, amplifying a neomycin resistance gene fragment by using a plasmid pMA5 as a template, digesting and connecting the plasmids and the fragments by using restriction endonuclease to obtain a recombinant plasmid, providing a repair element for a nuclease shearing site, carrying out enzyme digestion on the plasmids, carrying out gel recovery on the fragments containing a repair sequence and an antibiotic marker, connecting the fragments into pHY-Cas9-sgRNA scaffold in the step (3), and completing construction of a Bacillus licheniformis amylase coding gene editing plasmid.
A Bacillus licheniformis genome editing system based on a CRISPR-Cas9 system and containing the Bacillus licheniformis genome editing vector based on the CRISPR-Cas9 system is prepared by the following steps: and (3) electrically transforming the Bacillus licheniformis genome editing vector based on the CRISPR-Cas9 system into an electrically transformed induction state Bacillus licheniformis cell, and then culturing to obtain the Bacillus licheniformis genome editing system.
The electrotransformation competent Bacillus licheniformis is 9945a electrotransformation competent cell.
The preparation method of the bacillus licheniformis 9945a electrotransformation competent cell comprises the following steps:
(1) inoculating the bacillus licheniformis 9945a into 20mL LB culture medium, culturing at 37 ℃ and 180r/min overnight;
(2) transferring 2mL of the overnight culture into 50mL of electro-transformation growth medium (LB +0.5M sorbitol) of Bacillus licheniformis at 250r/min at 37 ℃ to OD600 of 0.85-0.95, and placing the shake flask in an ice bath for 10 min;
(3) centrifuging for 5min at 5000g, collecting thallus, washing thallus with electrotransformation washing culture medium (0.5M sorbitol +0.5M mannitol + 10% glycerol) of Bacillus licheniformis for 4 times, suspending thallus with 0.8mL of washing culture medium, and packaging into 80 μ L centrifuge tube of 1.5 mL.
The beneficial technical effects of the invention are as follows:
all elements required by the system, the sgRNA mediated by a strong promoter, the homologous repair sequence and the endonuclease Cas9 are integrated in the same recombinant plasmid, after the recombinant plasmid is transformed into a bacillus licheniformis cell, the sgRNA can recognize a specific region on a genome and guide the Cas9 protein to be combined to a target site, and under the action of the protein, a double-strand break gap is formed at the target site, and most cells die; the homologous repair sequence introduced with the recombinant plasmid is integrated to the gap through double exchange under the action of recombinase, so that cells successfully repaired by gene homology survive, and artificially designed genotypes such as gene inactivation, foreign gene insertion and the like are formed.
The gene knockout efficiencies of the CRISPR/Cas9 system constructed by the invention for three target sites of the same gene are respectively 58%, 39% and 37%, and the CRISPR/Cas9 system is more convenient and efficient compared with the existing Bacillus licheniformis gene editing mode.
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FIG. 1 is a schematic representation of the starting vector pHY-xyl;
FIG. 2 is a schematic representation of pHY-Cas9-sgRNA scaffold.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
Example 1
A preparation method of a Bacillus licheniformis genome editing vector based on a CRISPR-Cas9 system, comprising the following steps:
(1) designing and synthesizing a Cas9 protein expression frame (SEQ ID NO.1), a sgRNA scaffold expression frame (SEQ ID NO.2) and a plurality of sgRNA fragments (SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8) which accord with the codon characteristics of the bacillus licheniformis;
extracting the genomic DNA of the bacillus licheniformis 9945a, and amplifying by using pfu DNA polymerase and primers P1(5'-GCCAAGCTTTTACGACCTTTATGATTTAG-3') and P2(5'-CGAAAGCTTATTTGCAACCGAGCTGTCGC-3') to obtain an amylase encoding gene part open reading frame truncamyL;
reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min and then the next cycle: denaturation at 95 ℃ for 10s, annealing at 54 ℃ for 10s, extension at 72 ℃ for 1min, 30 cycles; extending for 10min at 72 ℃, and keeping the temperature at 4 ℃.
The amplified product is cloned to pMD18-T-simple vector after being purified, and plasmid T-truncamyL is obtained. And then, using primers P3(5'-GCCGAATTCAAAAGGATTGAAGGATGCTT-3') and P4(5'-ATAGTCGACGTTAATGCGCCATGACAGCC-3') to amplify the neomycin resistance gene fragment neo by using a plasmid pMA5 as a template, digesting and connecting the plasmids and the fragments by using restriction enzymes KpnI and SalI to obtain a recombinant plasmid T-truncamy L-neo, providing a repair element for a nuclease shearing site, performing single digestion on the plasmids by using HindIII, and performing gel recovery on a fragment amyL-neo containing a repair sequence and an antibiotic marker for later use.
(2) Cloning the Cas9 protein expression frame into an enzyme-cut starting vector pHY xyl (SEQ ID NO.9) to obtain pHY-Cas 9;
using pMA5 plasmid as a template, using primers P5(5'-GCCAAGCTTTTTTGAGTGATCTTCTCAAA-3') and P6(5'-ACGTCTAGACGCTCCTTTTTAGGTGGCAC-3') to amplify to obtain a bacillus promoter pHpaII, using HindIII and XbaI to double-enzyme-cut an amplification product, inserting the amplification product into a corresponding site of a shuttle plasmid pHY-B.S.xyl to obtain a plasmid pHY-B.S.xyl-pHpaII, using primers P7(5'-CGGTCTAGAAGTATTAAGTATTGTTTTAT-3') and P8(5'-CGAAGATCTAAAAAAAGCACCGACTCGGT-3') to amplify from a plasmid pCas9 to obtain tracrRNA, processing by XbaI and BglII, and connecting the tracrRNA into the corresponding site of the plasmid pHY-B.S.xyl-pHpaII to obtain a recombinant plasmid pHY-B.S.xyl-pHpaII-tracrRNA;
this recombinant plasmid was single digested with HindIII and ligated into the previously obtained fragment amyL-neo to give plasmid pHY-B.S.xyl-pHpaII-tracrRNA-amyL-neo (pHY-Cas 9).
(3) Cloning the sgRNA scaffold expression frame into the pHY-Cas9 prepared in the step (2) to obtain pHY-Cas9-sgRNA scaffold;
the plasmid pCas9 is used as a template, primers P9(5'-CGGAGATCTATGGATAAGAAATACTCAAT-3') and P10(5'-AGACCCGGGTCAGTCACCTCCTAGCTGAC-3') are used for amplifying an endonuclease encoding gene fragment Cas9, and the plasmid pHY-B.S.xyl-pHpaII-tracrRNA-amyL-neo BamHI and SmaI sites are ligated into the BamHI and SmaI sites of the plasmid pHY-B.S.xyl-pHpaII-tracrRNA-amyL-neo after double enzyme digestion, so that the construction of a CRISPR/Cas9 mediated Bacillus licheniformis gene knockout plasmid pHY-Cas9-sgRNA scaffold is completed.
(4) And cloning any one of a plurality of sgRNA fragments which are designed and synthesized by taking amyL as a target site into pHY-Cas9-sgRNA scaffold to obtain the bacillus licheniformis genome editing vector.
A Bacillus licheniformis genome editing system based on a CRISPR-Cas9 system and containing the Bacillus licheniformis genome editing vector based on the CRISPR-Cas9 system is prepared by the following steps: and (3) electrically transforming the Bacillus licheniformis genome editing vector based on the CRISPR-Cas9 system into an electrically transformed induction state Bacillus licheniformis cell, and then culturing to obtain the Bacillus licheniformis genome editing system.
(1) Adding plasmid DNA into the prepared bacillus licheniformis competent cells; transferring the cells into a 0.2cm electric transformation cup, standing on ice for 3-5min, shocking with an eppendorf electric transformation instrument at 2100V for 1 time, and rapidly adding 900ul of an electric transformation recovery culture medium (LB +0.5M sorbitol +0.38M mannitol) of Bacillus licheniformis after shocking;
(2) restoring and culturing at 37 ℃ for 3h at 100r/min, coating a tetracycline resistant plate containing 10 mu g/mL, and culturing at 37 ℃ for 20h to verify the grown transformant;
(3) the obtained recombinant Bacillus licheniformis positive transformant carrying the gene knockout plasmid is inoculated into LBT culture medium for overnight culture to serve as a seed, the inoculation amount of the transformant is 1 percent, the transformant is inoculated into 30mL of fresh LBT culture medium, the culture is carried out for 6h in a 250mL triangular flask at 37 ℃ under the condition of 250r/min, and then, xylose is added to the culture medium until the final concentration is 10g/L, and the culture is continued for 24 h. Then inoculating the culture solution into a culture medium containing 10g/L xylose for subculture for 12h by using the inoculation amount of 1%, repeating for 2 times, finally coating 200 mu L of the culture solution on an LBT (local binding site) plate added with 6 mu g/mL neomycin, and performing static culture at 37 ℃ for 24 h;
(4) carrying out colony PCR detection by using specific primers P11(5'-GCGGATGTGGGCTACGG-3') and P12(5'-ATTAATGCCGCCAAACC-3') aiming at upstream/downstream regions of homologous arms on a genome, carrying out PCR detection on transformants with changed amyL gene fragment sizes, recovering amplified fragments, sending the amplified fragments to Nanjing Jinzhi Biotech company for sequencing, comparing sequencing results with original genes, and detecting whether the amyL genes are knocked out successfully;
(5) 1mL of soluble starch (1%, w/v) is mixed with 0.25mL of citric acid-Na 2HPO4 buffer solution (0.2mol/L, pH5.0), 0.1mL of α -amylase solution is added after the temperature bath is carried out for 5min at 50 ℃, 0.1mL of HCl solution (0.1mol/L) is immediately added after the temperature is continuously kept for 10min to terminate the reaction, finally, the reducing sugar in the reaction solution is quantified, the maltose dried to constant weight is taken as a standard sample to draw a DNS standard curve, and one α -amylase activity unit (U) is defined as the enzyme amount required for generating 1mg of maltose per minute under the reaction conditions.
The present invention is described in detail in order to make those skilled in the art understand the content of the present invention and implement the same, and the present invention is not limited to the above embodiments, and all equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (6)
1. Bacillus licheniformis (based on CRISPR-Cas9 systemBacillus licheniformis) The genome editing vector is characterized in that the preparation method of the Bacillus licheniformis genome editing vector comprises the following steps: integrating a Cas9 protein expression frame and a sgRNA scaffold expression frame in a starting vector pHY xyl to obtain pHY-Cas9-sgRNA scaffold, and then integrating any one of a plurality of sgRNA fragments which are designed and synthesized by taking amyL as a target site into the pHY-Cas9-sgRNA to prepare the bacillus licheniformis genome editing vector;
the Cas9 protein expression cassette includes PxylPromoter, Cas9 protein and amyL terminator;
the sequence of the Cas9 protein expression box is shown as SEQ ID NO. 1;
the sequence of the starting vector pHY-xyl is shown in SEQ ID NO. 9.
2. A method for preparing a bacillus licheniformis genome editing vector based on CRISPR-Cas9 system according to claim 1, comprising the following steps:
(1) designing and synthesizing a Cas9 protein expression frame, a sgRNA scaffold expression frame and a plurality of sgRNA fragments which accord with the codon characteristics of the bacillus licheniformis;
(2) cloning the Cas9 protein expression frame into an enzyme-cut starting vector pHY xyl to obtain pHY-Cas 9;
(3) cloning the sgRNA scaffold expression frame into the pHY-Cas9 prepared in the step (2) to obtain pHY-Cas9-sgRNA scaffold;
(4) and cloning any one of a plurality of sgRNA fragments which are designed and synthesized by taking amyL as a target site into pHY-Cas9-sgRNA scaffold to obtain the bacillus licheniformis genome editing vector.
3. The Bacillus licheniformis genome editing vector according to claim 1, characterized in that the sgRNAscaffold expression cassette comprises PHpaIIPromoter and sgRNA scaffold.
4. A bacillus licheniformis genome editing vector according to claim 1, characterized in that the sgRNAscaffold expression cassette has the sequence shown in SEQ ID No. 2.
5. The bacillus licheniformis genome editing vector according to claim 1, characterized in that the sequence of the sgRNA fragment is shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.
6. A bacillus licheniformis genome editing system containing the bacillus licheniformis genome editing vector based on the CRISPR-Cas9 system according to claim 1, characterized in that the bacillus licheniformis genome editing system is prepared by the following steps: and (3) electrically transforming the Bacillus licheniformis genome editing vector based on the CRISPR-Cas9 system into a Bacillus licheniformis cell, and then culturing to obtain the Bacillus licheniformis genome editing system.
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| CN109022476B (en) * | 2018-07-16 | 2022-08-09 | 天津科技大学 | Bacillus licheniformis CRISPR-Cas9 gene editing system and application thereof |
| CN108929882B (en) * | 2018-08-06 | 2021-08-03 | 江南大学 | Gene editing method and application of bacillus licheniformis |
| CN109266676B (en) * | 2018-09-18 | 2020-12-11 | 华南农业大学 | Method for transforming Siamese bacillus by electric shock |
| BR112021018606A2 (en) | 2019-03-19 | 2021-11-23 | Harvard College | Methods and compositions for editing nucleotide sequences |
| DE112021002672T5 (en) | 2020-05-08 | 2023-04-13 | President And Fellows Of Harvard College | METHODS AND COMPOSITIONS FOR EDIT BOTH STRANDS SIMULTANEOUSLY OF A DOUBLE STRANDED NUCLEOTIDE TARGET SEQUENCE |
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