CN107400677A - A kind of bacillus licheniformis genome editor's carrier based on CRISPR Cas9 systems and preparation method thereof - Google Patents

A kind of bacillus licheniformis genome editor's carrier based on CRISPR Cas9 systems and preparation method thereof Download PDF

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CN107400677A
CN107400677A CN201710590555.XA CN201710590555A CN107400677A CN 107400677 A CN107400677 A CN 107400677A CN 201710590555 A CN201710590555 A CN 201710590555A CN 107400677 A CN107400677 A CN 107400677A
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石贵阳
李由然
张梁
丁重阳
顾正华
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Jiangnan University
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Abstract

The invention discloses a kind of bacillus licheniformis genome editor's carrier based on CRISPR Cas9 systems, the preparation method of bacillus licheniformis genome editor's carrier is：Cas9 protein expressions frame and sgRNA scaffold expression cassettes are incorporated into the carrier pHY xyl that set out, obtain pHY Cas9 sgRNA scaffold, then any one in a plurality of sgRNA fragments for synthesis being designed using amyL as target site is incorporated into described pHY Cas9 sgRNA scaffold, and bacillus licheniformis genome editor's carrier is made.The present invention can greatly improve the difficult present situation of current lichens bacillus gene editor, significantly improve the efficiency of gene editing.

Description

A kind of bacillus licheniformis genome editor's carrier based on CRISPR-Cas9 systems And preparation method thereof
Technical field
The present invention relates to gene technology field, more particularly, to it is a kind of be connected into specific sgRNA fragments based on Bacillus licheniformis genome editor's carrier of CRISPR-Cas9 systems.
Background technology
Bacillus licheniformis is a kind of Gram-positive bacillus, and it only has cell monolayer film, in drying, lacks the poles such as nutrition Endospore can be generated under the mal-condition of end, treats that condition is adapted to restoration ecosystem again.Bacillus licheniformis has wide variety of Industrial microorganism.It is not only the main producing strains of large industrial enzyme preparation-amylase and protease；Simultaneously because its is excellent Secretion capacity, the characteristic of simple fermentation condition and food security is used for the expressive host of foreign gene again, and production is a variety of Food enzyme preparation.With the complete parsing of its type strain ATCC14580 genomes, people are to this kind of strain growth and generation The understanding lifting thanked has opened up new application for this industrial microorganism in turn to a new aspect.
Currently using Genomic sequence information as blueprint, based on engineer heredity disturbance and carry out metabolic engineering into The most effective strategy of phenotype is needed for the complicated metabolic pathway of research bacillus licheniformis and progress strain transformation structure.People For most genetic modification instruments for developing of editor's bacterial genomes for bacillus licheniformis genetic manipulation all by It is effective to prove, however still suffer from some limitations have impact on these instruments in bacillus licheniformis in this special host should With the raising of efficiency.Such as except the Homologous integration double crossing over method that traditional use PCR fragment (mutation box) mediates carries out gene Beyond editor, also a kind of gene knockout method that (allelic exchange) is replaced based on genome equipotential, this method is most Big feature is the knockout of target gene and integration is completed under Negative selection mark-mazF auxiliary.MazF genes Induced expression in Escherichia coli can lethal cell so as to be screened, this method is sensitive compared with the screening that antibiotic marker aids in Degree and the degree of accuracy greatly improve.However, this system still relies on antibiotic marker in operation realizes integration mutation The screening of box；, will be on genome although FLP recombinases can be used to delete the antibiotic marker in subsequent experimental in theory Leave obvious " scar ".In addition, the function of FLP recombinases significant difference in different hosts, significantly limit this base Because of the versatility of edit methods.
The short palindrome in rule cluster interval repeats (the Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR) and CRISPR mediation gene-splicing system (CRISPR-associated, Cas) be a kind of generally existing in bacterium and archeobacteria immune system, it with efficient identification and can be cut into all of cell Such as bacteriophage or plasmid exogenous DNA.Its mode of action is as follows：Short dna sequence group between the repetition of the CRISPR palindrome Into the CRISPR arrays (CRISPR array) of target gene, this array is transcribed and processed between repetitive sequence, shape Into CRISPR-RNA (crRNA).Then crRNA guides Cas nucleases and navigates to target site, in specific PAM The shearing to target sequence is completed under the auxiliary of (Protospacer-adjacent Motif) sequence.Come from streptococcus pyogenes The natural CRISPR/Cas9 systems of (Streptococcus pyogenes) are contained by crRNA and trans regulation and control activation crRNA (tracrRNA) the double RNA complexs formed, this complex can mediate the effect of Cas9 endonucleases, and this system is people Study one of earliest CRISPR/Cas9 systems.Recently, the system has been successfully applied to the gene volume of multiple-microorganism Volume, including the Escherichia coli of Gram-negative bacteria and the S.pneumonia of gram-positive bacteria and Lactobacillus Reuteri, it is quick to bacterial genomes as a result to show that this system can be realized, easily directed modification.
But the bacillus licheniformis genome editing system for being currently based on CRISPR-Cas9 systems is not yet built so that This advanced technology can not be played in bacillus licheniformis this essential industry microorganism.
The content of the invention
In view of the above-mentioned problems existing in the prior art, the applicant provides one kind to be based on CRISPR-Cas9 systems Bacillus licheniformis genome editor's carrier and preparation method thereof.The present invention can greatly improve current bacillus licheniformis base Because editing difficult present situation, the efficiency of gene editing is significantly improved.
Technical scheme is as follows：
A kind of bacillus licheniformis genome editor's carrier based on CRISPR-Cas9 systems, the bacillus licheniformis The preparation method of genome editor's carrier is：Cas9 protein expressions frame and sgRNA scaffold expression cassettes are incorporated into load of setting out In body pHY xyl, pHY-Cas9-sgRNA scaffold are obtained, then will design a plurality of of synthesis by target site of amyL Any one in sgRNA fragments is incorporated into described pHY-Cas9-sgRNA scaffold, and the lichens gemma bar is made Bacterium genome editor's carrier.
The Cas9 protein expressions frame includes PxylPromoter, Cas9 albumen and amyL terminators.
The sequence of the Cas9 protein expressions frame is as shown in SEQ ID NO.1.
The gRNA scaffold expression cassettes include PHpaIIPromoter and sgRNA scaffold.
The sequence of the gRNA scaffold expression cassettes is as shown in SEQ ID NO.2.
The sequence of the sgRNA fragments such as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, Shown in SEQ ID NO.7 or SEQ ID NO.8.
The sequence of the carrier pHY-xyl that sets out is as shown in SEQ ID NO.9.
A kind of preparation method of bacillus licheniformis genome editor's carrier based on CRISPR-Cas9 systems, the system Preparation Method comprises the following steps：
(1) Cas9 protein expressions frame, the sgRNA for meeting bacillus licheniformis password sub-feature are designed and synthesized Scaffold expression cassettes and a plurality of sgRNA fragments；
The encoding gene of Cas9 albumen is optimized according to the codon preference of bacillus licheniformis and synthesized in vitro, according to According to the appropriate targets synthesis sgRNA scaffold expression cassettes inside target gene amyL expression cassettes and a plurality of sgRNA fragments；
(2) Cas9 protein expression frames are cloned into the carrier pHY xyl that set out through digestion, obtain pHY-Cas9；
The Cas9 protein coding genes that codon preference in step (1) optimizes are cloned into the carrier pHY-xyl that sets out In, pHY-Cas9 is obtained, Cas9 protein coding genes is expressed under the mediation of xylose evoked promoter；
(3) sgRNA scaffold expression cassettes are cloned into pHY-Cas9 made from step (2), obtain pHY-Cas9- sgRNA scaffold；
Using pMA5 plasmids as template, expanded using primer and obtain promoter, plasmid pHY- is inserted after amplified production digestion Cas9, then the sgRNA scaffold expression cassettes of synthesis in step (1) are connected into the corresponding site into above-mentioned plasmid after digestion, Obtain pHY-Cas9-sgRNA scaffold；
(4) any one in a plurality of sgRNA fragments for synthesis being designed using amyL as target site is cloned into pHY-Cas9- In sgRNA scaffold, reconnect and into gene editing repairing sequence, obtain described bacillus licheniformis genome editor load Body；
Bacillus licheniformis genomic DNA is extracted, target gene fractional open is obtained using archaeal dna polymerase and primer amplification Reading frame truncamyL, T-A is cloned into pMD18-T-simple carriers to amplified production after purification, reuses and regards it as plasmid pMA5 For template amplification neomycin resistance gene fragment, above plasmid and fragment using digestion with restriction enzyme and connect, and obtain Recombinant plasmid, provides for nuclease shearing site and repairs element, digestion above plasmid, and glue reclaim includes repairing sequence and antibiotic The fragment of mark, the fragment is connected into the pHY-Cas9-sgRNA scaffold in step (3), completes bacillus licheniformis and form sediment The structure of powder enzyme coding gene editor's plasmid.
It is a kind of containing bacillus licheniformis genome editor's carrier based on CRISPR-Cas9 systems based on The bacillus licheniformis genome editing system of CRISPR-Cas9 systems, the bacillus licheniformis genome editing system Preparation method is：By the bacillus licheniformis genome editor carrier electricity conversion based on CRISPR-Cas9 systems to electricity conversion sense Answer in state Bacillus licheniformis cell, then cultivated to obtain described bacillus licheniformis genome editing system.
The electric transformed competence colibacillus bacillus licheniformis is 9945a Electroporation-competent cells.
The bacillus licheniformis 9945a Electroporation-competent cells preparation methods：
(1) bacillus licheniformis 9945a is inoculated with 20mL LB culture mediums, and 37 DEG C, 180r/min is incubated overnight；
(2) overnight culture 2mL is forwarded to electricity conversion growth medium (the LB+0.5M sorbs of 50mL bacillus licheniformis Alcohol) in, 250r/min, 37 DEG C to OD600 are 0.85-0.95, and shaking flask is put into ice bath 10min；
(3) 5000g collects thalline after centrifuging 5min, with electricity conversion washing culture medium (the 0.5M sorbs of bacillus licheniformis The glycerine of alcohol+0.5M mannitol+10%) washing thalline 4 times, finally wash culture medium suspension thalline with 0.8mL, 80 μ L of packing in Complete to prepare in 1.5mL centrifuge tubes.
The present invention is beneficial to be had technical effect that：
All elements needed for present system, the sgRNA of strong promoter mediation, homologous repairing sequence and endonuclease Enzyme Cas9, it is integrated in same recombinant plasmid, after the inverted entrance Bacillus licheniformis cell of recombinant plasmid, sgRNA can know Specific region on other genome simultaneously guides Cas9 protein bindings to target site, in the presence of the albumen, at target site The breach of a double-strand break is formed, most of cell is thus dead；The homologous repairing sequence introduced with recombinant plasmid exists Indentation, there is integrated into by double crossing in the presence of recombinase, the successful cell of DNA homolog reparation survives with regard to this, and shape Into the genotype of the engineer such as gene inactivation and foreign gene insertion.
CRISPR/Cas9 systems constructed by the present invention are directed to the gene knockout efficiency of three target sites of same gene Respectively 58%, 39% and 37%, it is more convenient and efficient compared with existing bacillus licheniformis gene editing mode.
Brief description of the drawings
Fig. 1 is the carrier pHY-xyl schematic diagram of setting out；
Fig. 2 is pHY-Cas9-sgRNA scaffold schematic diagram.
Embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
Embodiment 1
A kind of preparation method of bacillus licheniformis genome editor's carrier based on CRISPR-Cas9 systems, the system Preparation Method comprises the following steps：
(1) design and synthesize meet bacillus licheniformis password sub-feature Cas9 protein expressions frame (SEQ ID NO.1), SgRNA scaffold expression cassettes (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、SEQ ID NO.8)；
Bacillus licheniformis 9945a genomic DNAs are extracted, use pfu archaeal dna polymerases and primer P1 (5'- GCCAAGCTTTTACGACCTTTATGATTTAG-3') expand with P2 (5'-CGAAAGCTTATTTGCAACCGAGCTGTCGC-3') Increase and obtain amylase encoding gene fractional open reading frame truncamyL；
Reaction condition：Enter subsequent cycle after 94 DEG C of pre-degeneration 5min：95 DEG C of denaturation 10s, 54 DEG C of annealing 10s, 72 DEG C are prolonged Stretch 1min, 30 circulations；72 DEG C of extension 10min, 4 DEG C of insulations.
T-A is cloned into pMD18-T-simple carriers to amplified production after purification, obtains plasmid T-truncamyL.Reuse Primer P3 (5'-GCCGAATTCAAAAGGATTGAAGGATGCTT-3') and P4 (5'- ATAGTCGACGTTAATGCGCCATGACAGCC-3') using plasmid pMA5 as template amplification neomycin resistance gene fragment neo, with Upper plasmid and fragment are digested and connected using restriction enzyme KpnI and SalI, obtain recombinant plasmid T-truncamyL- Neo, provided for nuclease shearing site and repair element, with HindIII single endonuclease digestion above plasmids, glue reclaim include repairing sequence and The fragment amyL-neo of antibiotic marker is stand-by.
(2) Cas9 protein expression frames are cloned into the carrier pHY xyl that set out (SEQ ID NO.9) through digestion, obtained pHY-Cas9；
Using pMA5 plasmids as template, primer P5 (5'-GCCAAGCTTTTTTGAGTGATCTTCTCAAA-3') and P6 is used (5'-ACGTCTAGACGCTCCTTTTTAGGTGGCAC-3') amplification obtains bacillus promoter pHpaII, and amplified production makes With the corresponding site that shuttle plasmid pHY-B.S.xyl is inserted after HindIII and XbaI double digestions, plasmid pHY-B.S.xyl- is obtained PHpaII, with primer P7 (5'-CGGTCTAGAAGTATTAAGTATTGTTTTAT-3') and P8 (5'- CGAAGATCTAAAAAAAGCACCGACTCGGT-3') amplification obtains tracrRNA from plasmid pCas9, through XbaI and BglII The corresponding site into plasmid pHY-B.S.xyl-pHpaII is connected after processing, obtains recombinant plasmid pHY-B.S.xyl-pHpaII- tracrRNA；
By this recombinant plasmid HindIII single endonuclease digestions, connect into the fragment amyL-neo obtained before, obtain plasmid pHY-B.S.xyl-pHpaII-tracrRNA-amyL-neo(pHY-Cas9)。
(3) sgRNA scaffold expression cassettes are cloned into pHY-Cas9 made from step (2), obtain pHY-Cas9- sgRNA scaffold；
Using plasmid pCas9 as template, using primer P9 (5'-CGGAGATCTATGGATAAGAAATACTCAAT-3') and P10 (5'-AGACCCGGGTCAGTCACCTCCTAGCTGAC-3') amplification of nucleic acid restriction endonuclease encoding gene segment Cas9, warp Connected after BglII and SmaI double digestions into plasmid pHY-B.S.xyl-pHpaII-tracrRNA-amyL-neo BamHI and SmaI sites, complete the bacillus licheniformis gene knockout plasmid pHY-Cas9-sgRNA scaffold of CRISPR/Cas9 mediations Structure.
(4) any one in a plurality of sgRNA fragments for synthesis being designed using amyL as target site is cloned into pHY-Cas9- In sgRNA scaffold, described bacillus licheniformis genome editor's carrier is obtained.
It is a kind of containing bacillus licheniformis genome editor's carrier based on CRISPR-Cas9 systems based on The bacillus licheniformis genome editing system of CRISPR-Cas9 systems, the bacillus licheniformis genome editing system Preparation method is：By the bacillus licheniformis genome editor carrier electricity conversion based on CRISPR-Cas9 systems to electricity conversion sense Answer in state Bacillus licheniformis cell, then cultivated to obtain described bacillus licheniformis genome editing system.
(1) DNA is added in the bacillus licheniformis competent cell of preparation；Cell is gone into 0.2cm electricity conversions In cup, 3-5min is placed on ice, is shocked by electricity 1 time with eppendorf electricity conversion instruments 2100V, 900ul lichens buds are rapidly added after electric shock The electricity conversion recovery media (LB+0.5M sorbierite+0.38M mannitol) of born of the same parents bacillus；
100r/min renewal cultivation 3h at (2) 37 DEG C, apply the tetracyclin resistance flat board containing 10 μ g/mL, 37 DEG C of culture 20h The transformant grown is verified afterwards；
(3) the restructuring bacillus licheniformis positive transformant for carrying gene knockout plasmid of acquisition is seeded to LBT trainings Foster base is incubated overnight as seed, is inoculated in 1% inoculum concentration equipped with LBT culture mediums fresh 30mL, in 250mL triangles In 37 DEG C in bottle, 6h is cultivated then under the conditions of 250r/min, xylose is added and continues to cultivate 24h to final concentration of 10g/L.Then, Above nutrient solution is seeded to the culture medium Secondary Culture 12h containing 10g/L xyloses with 1% inoculum concentration again, is repeated 2 times, most 200 μ L nutrient solutions are taken to be coated on the LBT flat boards of 6 μ g/mL neomycins of addition afterwards, in 37 DEG C of quiescent culture 24h；
(4) with the specific primer P11 (5'- for homology arm upstream/downstream region on genome GCGGATGTGGGCTACGG-3') and P12 (5'-ATTAATGCCGCCAAACC-3') carries out bacterium colony PCR detections, and PCR is detected The transformant that amyL genetic fragment sizes are changed, reclaim its amplified fragments and deliver to Nanjing Jin Weizhi biotechnologies company It is sequenced, sequencing result and protogene is compared, whether detection amyL genes knocks out success；
(5) 1mL soluble starches (1%, w/v) and 0.25mL citric acid-Na2HPO4 buffer solutions (0.2mol/L, pH are taken 5.0) mix, 0.1mL alphalise starch enzyme liquids are added after 50 DEG C of warm bath 5min, continue to be incubated that to add 0.1mL HCl immediately after 10min molten Liquid (0.1mol/L) terminating reaction, is finally quantified to the reduced sugar in reaction solution, to be dried to the maltose of constant weight as mark Sample draws DNS standard curves；One alpha-amylase enzyme-activity unit (U) is defined as：Under the above-described reaction conditions, generation per minute Enzyme amount needed for 1mg maltose.
The present invention is described in detail above, its object is to allow the personage for being familiar with this art to understand this Present disclosure is simultaneously carried out, and it is not intended to limit the scope of the present invention, and the invention is not restricted to above-mentioned reality Apply example, the equivalent change or modification that all Spirit Essences according to the present invention are done, should all cover protection scope of the present invention it It is interior.

Claims (9)

  1. A kind of 1. bacillus licheniformis genome editor's carrier based on CRISPR-Cas9 systems, it is characterised in that the lichens The preparation method of bacillus gene group editor's carrier is：Cas9 protein expressions frame and sgRNA scaffold expression cassettes are integrated In the carrier pHY xyl that set out, pHY-Cas9-sgRNA scaffold are obtained, then will design synthesis by target site of amyL A plurality of sgRNA fragments in any one be incorporated into described pHY-Cas9-sgRNA scaffold, the lichens is made Bacillus gene group editor's carrier.
  2. 2. bacillus licheniformis genome editor's carrier according to claim 1, it is characterised in that the Cas9 albumen table Include P up to framexylPromoter, Cas9 albumen and amyL terminators.
  3. 3. bacillus licheniformis genome editor's carrier according to claim 1, it is characterised in that the Cas9 albumen table Up to frame sequence as shown in SEQ ID NO.1.
  4. 4. bacillus licheniformis genome editor's carrier according to claim 1, it is characterised in that the gRNA Scaffold expression cassettes include PHpaIIPromoter and sgRNA scaffold.
  5. 5. bacillus licheniformis genome editor's carrier according to claim 1, it is characterised in that the gRNA The sequence of scaffold expression cassettes is as shown in SEQ ID NO.2.
  6. 6. bacillus licheniformis genome editor's carrier according to claim 1, it is characterised in that the sgRNA fragments Sequence such as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID Shown in NO.8.
  7. 7. bacillus licheniformis genome editor's carrier according to claim 1, it is characterised in that the carrier that sets out PHY-xyl sequence is as shown in SEQ ID NO.9.
  8. 8. a kind of preparation method of bacillus licheniformis genome editor's carrier based on CRISPR-Cas9 systems, its feature exist Comprise the following steps in the preparation method：
    (1) the Cas9 protein expressions frame for meeting bacillus licheniformis password sub-feature, sgRNAscaffold expression are designed and synthesized Frame and a plurality of sgRNA fragments；
    (2) Cas9 protein expression frames are cloned into the carrier pHY xyl that set out through digestion, obtain pHY-Cas9；
    (3) sgRNA scaffold expression cassettes are cloned into pHY-Cas9 made from step (2), obtain pHY-Cas9-sgRNA scaffold；
    (4) any one in a plurality of sgRNA fragments for synthesis being designed using amyL as target site is cloned into pHY-Cas9- In sgRNA scaffold, described bacillus licheniformis genome editor's carrier is obtained.
  9. 9. a kind of compile containing the bacillus licheniformis genome based on CRISPR-Cas9 systems described in any one of claim 1~8 Collect the bacillus licheniformis genome editing system based on CRISPR-Cas9 systems of carrier, it is characterised in that the lichens bud The preparation method of spore bacillus gene group editing system is：Bacillus licheniformis genome based on CRISPR-Cas9 systems is compiled Carrier electricity conversion is collected into Bacillus licheniformis cell, is then cultivated to obtain described bacillus licheniformis genome editor System.
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CN109022476A (en) * 2018-07-16 2018-12-18 天津科技大学 A kind of bacillus licheniformis CRISPR-Cas9 gene editing system and its application
CN109266676A (en) * 2018-09-18 2019-01-25 华南农业大学 A kind of method of electroporated Siam bacillus
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