SHUTTLE VECTOR FOR EXPRESSION IN E. COLI AND BACILLI

Abstract

Disclosed herein is a shuttle vector for use in E. coli and Bacilli including a high copy replication origin functional in E. coli, a low to medium copy ORI functional in Bacilli, and a synthetic constitutive regulatory nucleic acid conferring reduced constitutive expression compared to a respective starting regulatory nucleic acid molecule in a bacterial cell.

Claims

1. A shuttle vector comprising a. a high copy replication origin (ORI) functional in Escherichia coli, b. a low to medium copy ORI functional in bacilli, and c. a synthetic constitutive regulatory nucleic acid, wherein said synthetic constitutive regulatory nucleic acid is operably linked to a coding region which, upon high expression, would burden the bacterium, leading to reduced growth rate or vigour of said bacterium.

2. The shuttle vector of claim 1, wherein said coding region encodes an enzyme selected from the group consisting of a TALEN, a homing endonuclease, a meganuclease and a CRISPR/Cas enzyme.

3. The shuttle vector of claim 2, wherein the synthetic constitutive regulatory nucleic acid confers expression in bacilli.

4. The shuttle vector of claim 1 wherein the starting regulatory nucleic acid molecule conferring constitutive expression in a bacterial cell is selected from the group consisting of a. SEQ ID NO: 28 and 29, b. a nucleic acid molecule comprising at least 20 consecutive base pairs identical to 20 consecutive base pairs of a sequence described by SEQ ID NOs: 28 or 29, c. a nucleic acid molecule having an identity of at least 90% over the entire length of a sequence described by SEQ ID NO: 28 or 29, d. a nucleic acid molecule hybridizing under high stringent conditions with a nucleic acid molecule of at least 20 consecutive base pairs of a nucleic acid molecule described by SEQ ID NO: 28 or 29 and e. a complement of any of the nucleic acid molecules as defined in a) to d).

5. The shuttle vector of claim 1 wherein the synthetic regulatory nucleic acid molecule is selected from the group consisting of a. a nucleic acid molecule having a sequence of SEQ ID NO 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, b. a nucleic acid molecule comprising at least 20 consecutive base pairs identical to 20 consecutive base pairs of a sequence described by SEQ ID NO: 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47 c. a nucleic acid molecule having an identity of at least 90% over the entire length to a sequence described by SEQ ID NO: 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47 d. a nucleic acid molecule hybridizing under high stringent conditions with a nucleic acid molecule of at least 20 consecutive base pairs of a nucleic acid molecule described by any of SEQ ID NO: 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47 and e. a complement of any of the nucleic acid molecules as defined in a) to d), wherein the sequences as defined in b) to e) are distinct from the respective starting nucleic acid molecule.

6. The shuttle vector of claim 5, wherein the sequences as defined in b) to e) comprise at least one insertion or deletion compared to the respective starting nucleic acid molecule.

7. A method for expression in a bacterium a coding region which, upon high expression, would burden the bacterium, leading to reduced growth rate or vigour of said bacterium, the method comprising introducing the shuttle vector of claim 1 into said bacterium wherein said coding region is functionally linked to said synthetic constitutive regulatory nucleic acid conferring reduced constitutive expression.

8. The method of claim 7 wherein the coding region is a protein necessary for genome editing.

9. The method of claim 5 wherein the coding region encodes an enzyme selected from the group consisting of a TALEN, a homing endonuclease, a meganuclease or a CRISPR/Cas enzyme.

10. The method of claim 7, wherein the bacterium is a gram-positive or gram-negative bacterium.

11. The method of claim 10, wherein the bacterium is of a class selected from the group consisting of the class of Bacilli and the class of Gammaproteobacteria.

12. The method of claim 11, wherein the bacterium is of a family selected from the group consisting of the family of Bacillaceae and the family of Enterobacteriaceae.

13. The method of claim 12, wherein the bacterium is of a genus selected from the group consisting of the genus Bacilli and the genus Escherichia.

14. The method of claim 13, wherein the bacterium is selected from the group consisting of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus methylotrophicus, Bacillus cereus Bacillus paralicheniformis, Bacillus subtilis, and Bacillus thuringiensis.

15. The method of claim 14, wherein the bacterium is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus.

16. The method of claim 15, wherein the bacterium is Bacillus licheniformis.

17. A system for expression of a coding region encoding a protein which expression would burden the bacterium, the system comprising the shuttle vector of claim 1 and a coding region heterologous to said constitutive regulatory nucleic acid conferring reduced constitutive expression compared to a respective starting regulatory nucleic acid molecule in a bacterial cell.

18. The system of claim 17 wherein the coding region encodes a protein necessary for genome editing.

19. The system of claim 17 wherein the coding region encodes an enzyme selected from the group consisting of a TALEN, a homing endonuclease, a meganuclease and a CRISPR/Cas enzyme.

20. The shuttle vector of claim 1, wherein said coding region encodes an enzyme selected from the group consisting of a Cas9 or Cas12a enzyme.

Description

FIGURES

[0172] FIG. 1

[0173] The plasmid map of the single CRISPR/Cas9 plasmid pCC009 is depicted. The plasmid pCC009 is a derivative of the plasmid pJOE8999.1 carrying the spacer for the amyB gene of Bacillus licheniformis and the DNA donor sequences HomA and HomB 5′ and 3′ of the amyB gene respectively. PmanP: promoter of the Bacillus subtilis manP gene, pUC ORI: high-copy origin of replication E.° coli, Kanamycin resistance gene functional in both Bacillus and E.° coli, rep pE194: fragment of plasmid pE194 conferring temperature-sensitive plasmid replication in Bacillus, PvanP: promoter driving expression of the spacer-sgRNA (crRNA repeat+'gRNA), TO terminator from lambda, t1 t2 terminators from the E.° coli rrnB gene, HomA and HomB: sequences 5′ and 3′ of the amyB gene fused together for gene deletion, Cas9: Cas9 endonuclease from S. pyogenes.

[0174] FIG. 2:

[0175] The sequence alignment of selected regions of the mutated promoter sequences is shown—referenced against nt 15 to nt.128 of promoter sequences PV4 (SEQ ID 028) and PV8 (SEQ ID 029). Within the reference promoter sequences for the PV4 (SEQ ID 028) and PV8 (SEQ ID 029) promoters, the −35 and the −10 regions, the transcriptional start sites (TSS) and the Shine Dalgarno sequence (SD) are depicted in italic letters and shaded in grey. Nucleotide deletions, insertions and mutations are depicted in bold letters.

[0176] FIG. 3

[0177] Single colonies were analyzed by colony-PCR for deletion of the amyB gene of Bacillus licheniformis with oligonucleotides SEQ ID 009 and SEQ ID 010 lying outside the homology regions used for gene deletion. The gene deletion efficiency of the amylase amyB gene of Bacillus licheniformis as the percentage of clones with inactivated amylase gene relative to total of 20 clones analyzed for each gene deletion construct is plotted for each gene deletion construct as indicated. A. depicts the relative deletion efficiency of deletion plasmids derived from PV4 promoter variants. B. depicts the relative deletion efficiency of deletion plasmids derived from PV8 promoter variants.

[0178] FIG. 4

[0179] A. the gene deletion efficiency of the hag gene of Bacillus licheniformis as the percentage of clones with inactivated hag gene relative to total of 20 clones analyzed is plotted for two deletion constructs and promoter variants respectively as indicated. The average of three independent experiments with standard deviation is shown. The gene deletion of the hag gene was analyzed by colony PCR with oligonucleotides SEQ ID 087 and SEQ ID 088 lying outside the homology regions used for gene deletion. B. depicts the relative mutation efficiency of two deletion constructs and promoter variants respectively for introduction of point mutations within the degU gene of Bacillus licheniformis as the percentage of clones with mutated degU gene relative to total of 20 clones analyzed. The average of three independent experiments with standard deviation is shown. The gene mutation of the degU gene was analyzed by colony PCR with oligonucleotides SEQ ID 089 and SEQ ID 090 lying outside the homology region used for the introduction of the gene mutation, following restriction of the PCR fragment by PstI to differentiate between native and mutated degU locus.

[0180] FIG. 5

[0181] A. the gene deletion efficiency of the amylase amyE gene of Bacillus subtilis as the percentage of clones with inactivated amyE gene relative to total of 20 clones analyzed is plotted for two deletion constructs and promoter variants respectively as indicated. The average of three independent experiments with standard deviation is shown. The gene deletion of the amyE gene was analyzed by colony PCR with oligonucleotides SEQ ID 091 and SEQ ID 092 lying outside the homology regions used for gene deletion. B. depicts the relative deletion efficiency of two deletion constructs and promoter variants respectively for deletion of the Subtilisin protease aprE gene of Bacillus subtilis as the percentage of clones with inactivated aprE gene relative to total of 20 clones analyzed. The average of three independent experiments with standard deviation is shown. The gene deletion of the aprE gene was analyzed by colony PCR with oligonucleotides SEQ ID 093 and SEQ ID 094 lying outside the homology regions used for gene deletion.

[0182] FIG. 6

[0183] A. the gene deletion efficiency of the vpr gene of Bacillus licheniformis as the percentage of clones with inactivated vpr gene relative to total of 20 clones analyzed is plotted for three deletion constructs and spacer variants respectively as indicated. The gene deletion of the vpr gene was analyzed by colony PCR with oligonucleotides SEQ ID 095 and SEQ ID 096 lying outside the homology regions used for gene deletion. B. depicts the relative deletion efficiency of three deletion constructs and spacer variants respectively for deletion of the epr gene of Bacillus licheniformis as the percentage of clones with inactivated epr gene relative to total of 20 clones analyzed. The gene deletion of the epr gene was analyzed by colony PCR with oligonucleotides SEQ ID 097 and SEQ ID 098 lying outside the homology regions used for gene deletion.

[0184] FIG. 7

[0185] The gene integration efficiency of the PaprE-GFPmut2 expression cassette replacing the amyB gene of Bacillus licheniformis as the percentage of clones with integrated PaprE-GFPmut2 expression cassette relative to total of 20 clones analyzed is plotted for two different Bacillus licheniformis strains Bli #005 and P308 respectively as indicated. The average of two independent experiments with standard deviation is shown The integration was analyzed by colony PCR with oligonucleotides SEQ ID 009 and SEQ ID 010 lying outside the homology regions used for gene integration.

[0186] FIG. 8

[0187] The gene deletion efficiencies of the sporulation genes sigE, sigF and spoIIE of Bacillus pumilus as the percentage of clones with inactivated sporulation genes relative to total of 20 clones for each sporulation gene analyzed is plotted as indicated. The gene deletions of the sigE, sigF and spoIIE genes were analyzed by colony PCR with oligonucleotides SEQ ID 099 and SEQ ID 100, SEQ ID 101 and SEQ ID 102 and SEQ ID 103 and SEQ ID 104 respectively lying outside the homology regions used for gene deletion.

EXAMPLES

[0188] Material and Methods

[0189] The following examples only serve to illustrate the invention. The numerous possible variations that are obvious to a person skilled in the art also fall within the scope of the invention.

[0190] Unless otherwise stated the following experiments have been performed by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering and fermentative production of chemical compounds by cultivation of microorganisms. See also Sambrook et al. (Sambrook, J. and Russell, D. W. Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2001) and Chmiel et al. (Bioprocesstechnik 1. Einfuhrung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991).

[0191] Electrocompetent Bacillus licheniformis cells and electroporation Transformation of DNA into Bacillus licheniformis strain DSM641 and ATCC53926 is performed via electroporation. Preparation of electrocompetent Bacillus licheniformis cells and transformation of DNA is performed as essentially described by Brigidi et al (Brigidi, P., Mateuzzi, D. (1991). Biotechnol. Techniques 5, 5) with the following modification: Upon transformation of DNA, cells are recovered in 1 ml LBSPG buffer and incubated for 60 min at 37° C. (Vehmaanpera J., 1989, FEMS Microbio. Lett., 61: 165-170) following plating on selective LB-agar plates.

[0192] In order to overcome the Bacillus licheniformis specific restriction modification system of Bacillus licheniformis strains DSM641 and ATCC53926, plasmid DNA is isolated from Ec #098 cells as described below. For transfer into Bacillus lichenformis restrictase knockout strains, plasmid DNA is isolated from E.° coli INV110 cells (Life technologies).

[0193] Electrocompetent Bacillus pumilus Cells and Electroporation

[0194] Transformation of DNA into Bacillus pumilus DSM14395 is performed via electroporation. Preparation of electrocompetent Bacillus pumilus DSM14395 cells and transformation of DNA is performed as described for Bacillus licheniformis cells.

[0195] In order to overcome the Bacillus pumilus specific restriction modification system plasmid DNA is isolated from E.° coli DH10B cells and plasmid DNA is in vitro methylated with whole cell extracts from Bacillus pumilus DSM14395 according to the method as described for Bacillus licheniformis in patent DE4005025.

[0196] Electrocompetent Bacillus subtilis Cells and Electroporation

[0197] Transformation of DNA into Bacillus subtilis ATCC6051a is performed via electroporation as described for Bacillus licheniformis and Bacillus pumilus respectively. Plasmid DNA isolated from E.° coli DH10B cells can be readily used for transfer into Bacillus subtilis.

[0198] Plasmid Isolation

[0199] Plasmid DNA was isolated from Bacillus and E.° coli cells by standard molecular biology methods described in (Sambrook, J. and Russell, D. W. Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2001) or the alkaline lysis method (Birnboim, H. C., Doly, J. (1979). Nucleic Acids Res 7(6): 1513-1523). Bacillus cells were in comparison to E.° coli treated with 10 mg/ml lysozyme for 30 min at 37 C prior to cell lysis.

[0200] Annealing of Oligonucleotides to Form Oligonucleotide-Duplexes.

[0201] Oligonucleotides were adjusted to a concentration of 100 μM in water. 5 μl of the forward and 5 μl of the corresponding reverse oligonucleotide were added to 90 μl 30 mM Hepes-buffer (pH 7.8). The reaction mixture was heated to 95° C. for 5 min following annealing by ramping from 95° C. to 4° C. with decreasing the temperature by 0.1° C./sec (Cobb, R. E., Wang, Y., & Zhao, H. (2015). High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synthetic Biology, 4(6), 723-728).

[0202] Molecular Biology Methods and Techniques

[0203] Standard methods in molecular biology not limited to cultivation of Bacillus and E.° coli microorganisms, electroporation of DNA, isolation of genomic and plasmid DNA, PCR reactions, cloning technologies were performed as essentially described by Sambrook and Ru-sell. (Sambrook, J. and Russell, D. W. Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2001.)

[0204] Strains

[0205] E.° coli strain Ec #098

[0206] E.° coli strain Ec #098 is an E.° coli INV110 strain (Life technologies) carrying the DNA-methyltransferase encoding expression plasmid pMDS003 WO2019016051.

[0207] Generation of Bacillus licheniformis Gene k.o Strains

[0208] For gene deletion in Bacillus licheniformis strains DSM641 and ATCC53926 (U.S. Pat. No. 5,352,604) and derivatives thereof deletion plasmids were transformed into E.° coli strain Ec #098 made competent according to the method of Chung (Chung, C. T., Niemela, S. L., and Miller, R. H. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. U.S.A 86, 2172-2175), following selection on LB-agar plates containing 100 μg/ml ampicillin and 30 μg/ml chloramphenicol at 37° C. Plasmid DNA was isolated from individual clones and used for subsequent transfer into Bacillus licheniformis strains. The isolated plasmid DNA carries the DNA methylation pattern of Bacillus licheniformis strains DSM641 and ATCC53926 respectively and is protected from degradation upon transfer into B. licheniformis.

[0209] B. licheniformis P304: deleted restriction endonuclease Electrocompetent Bacillus licheniformis DSM641 cells (U.S. Pat. No. 5,352,604) were prepared as described above and transformed with 1 μg of pDe1006 restrictase gene deletion plasmid isolated from E.° coli Ec #098 following plating on LB-agar plates containing 5 μg/ml erythromycin at 30° C.

[0210] The gene deletion procedure was performed as described in the following: Plasmid carrying Bacillus licheniformis cells were grown on LB-agar plates with 5 μg/ml erythromycin at 45° C. driving integration of the deletion plasmid via Campbell recombination into the chromosome with one of the homology regions of pDe1006 homologous to the sequences 5′ or 3′ of the aprE gene. Clones were picked and cultivated in LB-media without selection pressure at 45° C. for 6 hours, following plating on LB-agar plates with 5 μg/ml erythromycin at 30° C. Individual clones were picked and screened by colony-PCR analysis with oligonucleotides SEQ ID 014 and SEQ ID 015 for successful genomic deletion of the restrictase gene. Putative deletion positive individual clones were picked and taken through two consecutive overnight incubation in LB media without antibiotics at 45° C. to cure the plasmid and plated on LB-agar plates for overnight incubation at 37° C. Single clones were analyzed by colony PCR for successful genomic deletion of the restrictase gene. A single erythromycin-sensitive clone with the correct deleted restrictase gene was isolated and designated Bacillus licheniformis P304.

[0211] B. licheniformis P308: Deleted Poly-Gamma Glutamate Synthesis Genes

[0212] Electrocompetent Bacillus licheniformis P304 cells were prepared as described above and transformed with 1 μg of pDe1007 pga gene deletion plasmid isolated from E.° coli INV110 cells (Life technologies) following plating on LB-agar plates containing 5 μg/ml erythromycin at 30° C.

[0213] The gene deletion procedure was performed as described for the deletion of the restrictase gene.

[0214] The deletion of the pga genes was analyzed by PCR with oligonucleotides SEQ ID 017 and SEQ ID 018 The resulting Bacillus licheniformis strain with deleted pga synthesis genes was named Bacillus licheniformis P308.

[0215] B. licheniformis Bli #002: deleted aprE gene Electrocompetent Bacillus licheniformis ATCC53926 cells were prepared as described above and transformed with 1 μg of pDe1003 aprE gene deletion plasmid isolated from E.° coli Ec #098 following plating on LB-agar plates containing 5 μg/ml erythromycin at 30° C. The gene deletion procedure was performed as described for the deletion of the restrictase gene. The deletion of the aprE gene was analyzed by PCR with oligonucleotides SEQ ID 020 and SEQ ID 021 The resulting Bacillus licheniformis strain with deleted aprE gene was named Bli #002.

[0216] B. licheniformis Bli #005: deleted poly-gamma glutamate synthesis genes

[0217] The poly-gamma-glutamate synthesis genes were deleted in Bacillus licheniformis Bli #002 as described for the deletion of the pga genes in Bacillus licheniformis P304 with the difference that the pDe1007 plasmid was isolated from E.° coli Ec #098 cells. The resulting strain was named Bli #005.

[0218] Plasmids

[0219] pEC194RS—Bacillus temperature sensitive deletion plasmid.

[0220] The plasmid pE194 is PCR-amplified with oligonucleotides SEQ ID 001 and SEQ ID 002 with flanking PvuII sites, digested with restriction endonuclease PvuII and ligated into vector pCE1 digested with restriction enzyme SmaI. pCE1 is a pUC18 derivative, where the BsaI site within the ampicillin resistance gene has been removed by a silent mutation. The ligation mixture was transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37 C on LB-agar plates containing 100 μg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest. The resulting plasmid is named pEC194S.

[0221] The type-II-assembly mRFP cassette is PCR-amplified from plasmid pBSd141R (accession number: KY995200) (Radeck, J., Meyer, D., Lautenschlager, N., and Mascher, T. 2017. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7: 14134) with oligonucleotides SEQ ID 003 and SEQ ID 004, comprising additional nucleotides for the restriction site BamHI. The PCR fragment and pEC194S were restricted with restriction enzyme BamHI following ligation and transformation into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37 C on LB-agar plates containing 100 μg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest. The resulting plasmid pEC194RS carries the mRFP cassette with the open reading frame opposite to the reading frame of the erythromycin resistance gene.

[0222] pDe1003—aprE Gene Deletion Plasmid

[0223] The gene deletion plasmid for the aprE gene of Bacillus licheniformis was constructed with plasmid pEC194RS and the gene synthesis construct SEQ ID 019 comprising the genomic regions 5′ and 3′ of the aprE gene flanked by BsaI sites compatible to pEC194RS. The type-II-assembly with restriction endonuclease BsaI was performed as described (Radeck, J., Meyer, D., Lautenschlager, N., and Mascher, T. 2017. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7: 14134) and the reaction mixture subsequently transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37 C on LB-agar plates containing 100 μg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest. The resulting aprE deletion plasmid is named pDe1003.

[0224] pDe1006—Restrictase Gene Deletion Plasmid

[0225] The gene deletion plasmid for the restrictase gene (SEQ ID 012) of the restriction modification system of Bacillus licheniformis DSM641(SEQ ID 011) was constructed with plasmid pEC194RS and the gene synthesis construct SEQ ID 013 comprising the genomic regions 5′ and 3′ of the restrictase gene flanked by BsaI sites compatible to pEC194RS. The type-II-assembly with restriction endonuclease BsaI was performed as described above and the reaction mixture subsequently transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37 C on LB-agar plates containing 100 μg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest. The resulting restrictase deletion plasmid is named pDe1006.

[0226] pDe1007—Poly-Gamma-Glutamate Synthesis Genes Deletion Plasmid

[0227] The deletion plasmid for deletion of the genes involved in poly-gamma-glutamate (pga) production, namely ywsC (pgsB), ywtA (pgsC), ywtB (pgsA), ywtC (pgsE) of Bacillus licheniformis was constructed as described for pDe1006, however the gene synthesis construct SEQ ID 016 comprising the genomic regions 5′ and 3′ flanking the ywsC, ywtA (pgsC), ywtB (pgsA), ywtC (pgsE) genes flanked by BsaI sites compatible to pEC194RS was used. The resulting pga deletion plasmid is named pDe1007.

[0228] Plasmid p689-T2A-Lac

[0229] The plasmid p689-T2A-lac comprises the lacZ-alpha gene flanked by BpiI restriction sites, again flanked 5′ by the T1 terminator of the E.° coli rrnB gene and 3′ by the T0 lambda terminator and was ordered as gene synthesis construct (SEQ ID 073).

[0230] Plasmid p890 PaprE-GFPmut2

[0231] The promoter of the aprE gene from Bacillus licheniformis of plasmid pCB56C (U.S. Pat. No. 5,352,604) was PCR-amplified with oligonucleotides SEQ ID 074 and SEQ ID 075. The GFPmut2 gene variant (accession number AF302837) with flanking BpiI restriction sites (SEQ ID 076) was ordered as gene synthesis fragment (Geneart Regensburg). The gene expression construct comprising the PaprE promoter from Bacillus licheniformis fused to the GFPmut2 variant was cloned into plasmid p689-T2A-lac by type-II-assembly with restriction endonuclease BpiI as described (Radeck, J., Meyer, D., Lautenschlager, N., and Mascher, T. 2017. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7: 14134) and the reaction mixture subsequently transformed into electrocompetent E.° coli DH10B cells. Transformants were spread and incubated overnight at 37° C. on LB-agar plates containing 100 μg/ml ampicillin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing. The resulting plasmid is named p890 PaprE-GFPmut2.

[0232] Plasmid pJOE8999.1:

[0233] Altenbuchner J. 2016. Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system. Appl Environ Microbiol 82:5421-5.

[0234] Plasmid pJOE-T2A

[0235] To allow for type-II-assembly (T2A) based one-step-cloning of the sgRNA and the homology regions for DSB repair the CRISPR/Cas9 plasmid pJOE8889.1 was modified as follows.

[0236] The type-II-assembly mRFP cassette from plasmid pBSd141R (accession number: KY995200) (Radeck, J., Meyer, D., Lautenschlager, N., and Mascher, T. 2017. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7: 14134) was modified such to remove multiple restriction sites and the BpiI restriction sites and ordered as gene synthesis fragment with flanking SfiI restriction sites (SEQ ID 005). The plasmid is named p #732. Plasmid p #732 and plasmid pJOE8999.1 were digested with SfiI (New England Biolabs, NEB) and the mRFP cassette of p #732 ligated into SfiI-digested pJOE8999.1 following transformation into competent E.° coli DH10B cells. Positive clones were screened on IPTG/X-Gal and kanamycin (20 μg/ml) containing LB agar plates for purple colonies (blue-white screening and mRFP1 expression). The resulting sequence-verified plasmid was named pJOE-T2A.

[0237] Plasmid pBW732

[0238] The 5′ homology region (also referred to as HomA) and the 3′ homology region (also referred to as HomB) adjacent to the amylase amyB gene of Bacillus licheniformis DSM641 was ordered as synthetic gene synthesis fragment with flanking XmaI restriction sites (SEQ ID 006). The plasmid pJOE8999.1 and the synthetic amyB-HomAB fragment are cleaved with restriction endonuclease XmaI following ligation with T4-DNA ligase (NEB) and transformation into electrocompetent E.° coli DH10B cells. The correct plasmid was recovered and named pBW732.

[0239] Plasmid pBW742

[0240] The 20 bp target sequence of the amyB gene for the sgRNA was designed using Geneious 11.1.5 (https://www.geneious.com). The resulting oligonucleotides SEQ ID 007 and Seq ID 008 with 5′ phosphorylation were annealed to form an oligonucleotide duplex. The CRISPR/Cas9 based gene deletion plasmid for the amyB gene of Bacillus licheniformis was constructed by type-II-assembly with restriction endonuclease BsaI as described (Radeck, J., Meyer, D., Lautenschlager, N., and Mascher, T. 2017. Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7: 14134) with the following components: pBW732 and the oligonucleotide duplex (SEQ ID 007, SEQ ID 008). The reaction mixture was transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37° C. on LB-agar plates containing 20 μg/ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing. The resulting amyB deletion plasmid is named pBW742.

[0241] T2A CRISPR Destination Vectors pCCO27 and pCCO28

[0242] Plasmid pCC014 and pCCO25 were modified such that region covering the spacer-sgRNA and amyB gene flanking homologous regions were replaced by the T2A cassette from plasmid pJOE-T2A. The backbones of pCC014 and pCCO25 were PCR amplified with oligonucleotides SEQ ID 050 and SEQ ID 051 and the T2A assembly cassette was PCR-amplified from pJOE-T2A with oligonucleotides SEQ ID 048 and SEQ ID 049 following PCR purification using the High Pure PCR purification Kit, digestion with DpnI and gel purification. The corresponding backbone PCR fragments and the T2A cassette PCR fragment were annealed in a 10 μl Gibson reaction following transformation into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37° C. on LB-agar plates containing 20 μg/ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing. The resulting pCC014 and pCCO25 derived T2A plasmid derivatives are designated pCCO27 and pCCO28 respectively.

[0243] pCCO29—Hag Gene Deletion Plasmid

[0244] The 20 bp target sequence of the hag gene for the sgRNA was designed using Geneious 11.1.5 as described before. The resulting oligonucleotides SEQ ID 056 and Seq ID 057 with 5′ phosphorylation were annealed to form an oligonucleotide duplex as described above.

[0245] The genomic regions 5′ and 3′ of the hag gene were PCR-amplified on genomic DNA from Bacillus licheniformis DSM641 with oligonucleotides SEQ ID 054 and Seq ID 053 and SEQ ID 052 and Seq ID 55 following fusion by overlap extension PCR with flanking oligonucleotides SEQ ID 053 and SEQ ID 054. The resulting PCR product was column purified (Qiagen PCR purification Kit). The CRISPR/Cas9 based gene deletion plasmid for the hag gene of Bacillus licheniformis was constructed by type-II-assembly with restriction endonuclease BsaI as described before with the following components: plasmid pCCO27 (PV4-5 promoter variant), the fused homology regions of the hag gene with flanking BsaI restriction sites and the oligonucleotide duplex (SEQ ID 056, SEQ ID 057). The reaction mixture was transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37° C. on LB-agar plates containing 20 μg/ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing. The resulting hag gene deletion plasmid is named pCCO29.

[0246] pCC030—Hag Gene Deletion Plasmid

[0247] The hag gene deletion construct was constructed as for pCCO29 however the plasmid pCCO28 (PV8-7 promoter variant) was used.

[0248] pCC031—degU32 Gene Editing Plasmid

[0249] The construction of the degU32 genome editing construct to introduce the degU H12L mutation was performed as for pCCO29 with the following modifications.

[0250] The degU32 homology regions introducing the mutations for the degU H12L mutation as well as the introduction of a silent point mutation to remove the PAM site were ordered as gene synthesis construct (Geneart, Regensburg) with flanking BsaI sites (SEQ ID 058). The 20 bp target sequence of the degU gene for the sgRNA was designed and the resulting oligonucleotides SEQ ID 059 and Seq ID060 with 5′ phosphorylation were annealed to form an oligonucleotide duplex as described above.

[0251] pCC032—degU32 Gene Editing Plasmid

[0252] The degU32 genome editing construct was made as described for pCC031 however the plasmid pCCO28 (PV8-7 promoter variant) was used.

[0253] pCC033—amyE Gene Deletion Plasmid

[0254] The fragment comprising the amyE spacer-sgRNA and homology regions of the 5′ and 3′ regions of the amyE gene from Bacillus subtilis was PCR-amplified from plasmid pCC004 (WO17186550) with oligonucleotides SEQ ID 061 and SEQ ID 062 with flanking BsaI restriction sites. The CRISPR/Cas9 based gene deletion plasmid for the amylase amyE gene was subsequently constructed by type-II-assembly with restriction endonuclease BsaI as described above with plasmid pCCO27 (PV4-5 promoter variant) and the PCR-amplified fragment. The reaction mixture was transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37° C. on LB-agar plates containing 20 μg/ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing. The resulting amyE gene deletion plasmid is named pCC033.

[0255] pCC034—amyE Gene Deletion Plasmid

[0256] The amyE gene deletion construct was constructed as for pCC033, however the plasmid pCCO28 (PV8-7 promoter variant) was used.

[0257] pCC035—aprE Gene Deletion Plasmid

[0258] The fragment comprising the aprE spacer (SEQ ID 064)-sgRNA and homology regions of the 5′ and 3′ regions of the aprE gene of Bacillus subtilis was ordered as synthetic gene fragment (SEQ ID 063) with flanking BsaI restriction sites. The CRISPR/Cas9 based gene deletion plasmid for the protease aprE gene was subsequently constructed by type-II-assembly with restriction endonuclease BsaI as described above with plasmid pCCO27 (PV4-5 promoter variant) and gene synthesis construct. The reaction mixture was transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37° C. on LB-agar plates containing 20 μg/ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing. The resulting aprE gene deletion plasmid is named pCC035.

[0259] pCC036—aprE Gene Deletion Plasmid

[0260] The aprE gene deletion construct was constructed as for pCC035, however the plasmid pCCO28 (PV8-7 promoter variant) was used.

[0261] pCC037—pCC039—Vpr Gene Deletion Plasmids

[0262] The CRISPR/Cas9 gene deletion constructs pCC037, pCC038 and pCC039 of the protease vpr gene of Bacillus licheniformis were constructed as described for pCC035, however with synthetic gene fragments comprising the vpr spacer-sgRNA and homology regions of the 5′ and 3′ regions of the vpr gene (SEQ ID 065). The resulting plasmids pCC037, pCC038 and pCC039 differ in the vpr spacer sequences (SEQ ID 066, SEQ ID 067, SEQ ID 068) within SEQ ID 065.

[0263] pCC040—pCC042—Epr Gene Deletion Plasmids

[0264] The CRISPR/Cas9 gene deletion constructs pCC040, pCC041 and pCC042 of the protease epr gene of Bacillus licheniformis were constructed as described for pCC035, however with synthetic gene fragments comprising the epr spacer-sgRNA and homology regions of the 5′ and 3′ regions of the epr gene (SEQ ID 069). The resulting plasmids pCC040, pCC041 and pCC042 differ in the epr spacer sequences (SEQ ID 070, SEQ ID 071, SEQ ID 072) within SEQ ID 069.

[0265] pCC043— GFP Gene Integration Plasmid

[0266] The 20 bp target sequence of the amyB gene for the sgRNA were ordered as oligonucleotides SEQ ID 007 and Seq ID 008 with 5′ phosphorylation following annealing to form an oligonucleotide duplex. The 5′ and 3′ regions of the amyB gene of Bacillus licheniformis were PCR-amplified with oligonucleotides SEQ ID 077 and SEQ ID 078 and SEQ ID 079 and SEQ ID 080 respectively.

[0267] The CRISPR/Cas9 based gene integration plasmid replacing the amyB gene of Bacillus licheniformis was constructed by type-II-assembly with restriction endonuclease BsaI as described as described above with the following components: pCCO27, the oligonucleotide duplex (SEQ ID 007, SEQ ID 008), the PCR-fragment of the 5′ homology region of the amyB gene, p890-PaprE-GFPmut2 and the PCR-fragment of the 3′ homology regions of the amyB gene. The reaction mixture was transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37° C. on LB-agar plates containing 20 μg/ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing. The resulting CRISPR/Cas9 based gene integration plasmid is named pCC043.

[0268] pCC044— sigE Gene Deletion Plasmid Bacillus pumilus

[0269] The CRISPR/Cas9 gene deletion construct pCC044 of the sigE gene of Bacillus pumilus DSM14395 was constructed as described for pCC035, however with a synthetic gene fragment (SEQ ID 082) comprising the sigE spacer (SEQ ID 081)-sgRNA and homology regions of the 5′ and 3′ regions of the sigE gene.

[0270] pCC045— sigF Gene Deletion Plasmid Bacillus pumilus

[0271] The CRISPR/Cas9 gene deletion construct pCC045 of the sigF gene of Bacillus pumilus DSM14395 was constructed as described for pCC035, however with a synthetic gene fragment (SEQ ID 084) comprising the sigF spacer (SEQ ID 083)-sgRNA and homology regions of the 5′ and 3′ regions of the sigF gene.

[0272] pCC046—spoIIE Gene Deletion Plasmid Bacillus pumilus

[0273] The CRISPR/Cas9 gene deletion construct pCC046 of the spoIIE gene of Bacillus pumilus DSM14395 was constructed as described for pCC035, however with a synthetic gene fragment (SEQ ID 086) comprising the spoIIE spacer (SEQ ID 085)-sgRNA and homology regions of the 5′ and 3′ regions of the spoIIE gene.

Example 1: Construction of CRISPR/Cas9 Genome Editing Plasmids with Constitutive Promoter

[0274] In order to introduce a constitutive promoter driving the expression of the Cas9 enzyme in plasmid pBW742 a two-step procedure was applied.

[0275] Frist, the t1t2t0 terminator (derived from pMUTIN) was introduced 5′ of the promoter PmanP of pBW742 to prevent potential read-through from the kanamycin selection marker.

[0276] The terminator sequence t1t2t0 was integrated into pBW742 upstream of the mannose promoter by Gibson assembly (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs). To this purpose, the terminator fragment (0.44 kb) was amplified by PCR with oligonucleotides SEQ ID 024 and SEQ ID 025 using pMutin2 (accession number AF072806) as the template. The corresponding vector backbone of pBW742 was amplified with oligonucleotides SEQ ID 022 and SEQ ID 023. The pBW742 amplicon was purified using the PCR product purification kit (Roche). After subsequent digestion of the pBW742 PCR product with DpnI (New England Biolabs), both PCR fragments were gel purified using the Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany) and annealed in a 1:2 ratio for 1 h at 50° C. E.° coli strain DH10B was transformed with the assembly reaction following plating on LB-agar plates containing 20 μg/ml kanamycin. Plasmid DNA was isolated from individual clones and analyzed for correctness by restriction digest and sequencing.

[0277] A deviation from the published reference sequence of pMutin2 was found. The SEQ ID 026 covers the part of the pMutin2 sequence, SEQ ID 027 covers the sequence deviation found within the corresponding region of pMutin2 found in the resulting plasmid pCC009.

[0278] Secondly, the mannose-inducible promoter PmanP was exchanged by two promoter variants of the constitutive promoter Pveg from Bacillus subtilis—namely PV4 and PV8—derived from Guiziou et al (Guiziou, S., V. Sauveplane, H. J. Chang, C. Clerte, N. Declerck, M. Jules, and J. Bonnet. 2016. A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Res. 44: 7495-7508). These promoter variants which comprise the Pveg promoter, a standardized TSS (transcriptional start site) region and the standardized ribosome binding site region RO, derived from the adapted Pveg promoter library that was screened on single copy level in Bacillus subtilis with regards to their altered expression levels. The PV4 and PV8 promoter sequences are listed as SEQ ID 028 and SEQ ID 029 respectively.

[0279] The integration of both promoter variants was carried out by Gibson assembly. Amplification of the PV4 and PV8 fragments was done stepwise. For both promoter fragments, using pCC009 as the template, oligonucleotides SEQ ID 024 and SEQ ID 030 were used for the first PCR (Phusion high fidelity DNA polymerase—NEB) and the resulting products served as the template for a second PCR with the oligonucleotides SEQ ID 024 and SEQ ID 031 for PV4 and SEQ ID 024 and SEQ ID 033 for PV8.

[0280] The vector backbone of pCC009 was PCR amplified using oligonucleotides SEQ ID 022 and SEQ ID 032. After purification of the vector amplicon with the PCR purification kit (Roche), PCR product digestion with DpnI was carried out to remove remaining circular plasmid DNA from the PCR reaction. Subsequently, the digested vector and both promoter fragments were purified using the Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany).

[0281] The vector amplicon of pCC009 was then annealed with the promoter fragments of PV4 and PV8, respectively, thereby replacing the mannose promoter PmanP with the PV4 and PV8 variants of the Pveg promoter.

[0282] The annealing reactions were subsequently transformed into E.° coli DH10B cells (Life technologies). Transformants were spread and incubated overnight at 37 C on LB-agar plates containing 20 μg/ml kanamycin. Plasmid DNA was isolated from 9 individual clones of PV4 promoter and 8 individual clones from promoter variant PV8 and analyzed for correctness by sequencing.

[0283] Table 1 summarizes the sequencing results of the various promoter variants: Analysis of clones from PV4-cloning reactions reveals that only sequences with point mutations, nucleotide insertions or deletions within the PV4 region could be recovered.

[0284] Analysis of clones from PV8-cloning reactions reveals that that only sequences with point mutations, nucleotide insertions or deletions within the PV8 region could be recovered. The resulting plasmids are summarized in Table 1.

TABLE-US-00005 TABLE 1 Plasmid Promoter variant SEQ ID pCC010 Pv4-1 034 pCC011 Pv4-2 035 pCC012 Pv4-3 036 pCC013 Pv4-4 036 pCC014 Pv4-5 037 pCC015 Pv4-6 038 pCC016 Pv4-7 039 pCC017 Pv4-8 040 pCC018 Pv4-9 039 pCC019 Pv8-1 041 pCC020 Pv8-2 042 pCC021 Pv8-3 043 pCC022 Pv8-4 044 pCC023 Pv8-5 045 pCC024 Pv8-6 041 pCC025 Pv8-7 046 pCC026 Pv8-8 047

[0285] Gene deletion efficiency of CRISPR/Cas9 based deletion plasmids Electrocompetent Bacillus licheniformis P308 cells were prepared as described above and transformed with 1 μg of amyB deletion plasmids pCC010-012, pCC014-017, pCC019-026 (with different promoter variants as depicted in Table 1) isolated from E.° coli INV110 cells (Life technologies) following plating on LB-agar plates containing 20 μg/ml kanamycin and incubation overnight at 37° C.

[0286] The next day 20 clones of each transformation reaction were subjected to colony-PCR to analyze for successful CRISPR/Cas9 based deletion of the amyB gene and with oligonucleotides SEQ ID 009 and SEQ ID 010, and further transferred onto fresh LB-agar plates without antibiotics following incubation at 48° C. overnight for plasmid curing.

[0287] The efficiency of amyB gene deletion for each CRISPR/Cas9 based deletion plasmid was calculated as the ratio in percentage of successful gene deletion based on the appearance of the expected smaller specific PCR-amplicon compared to the larger specific PCR-amplicon of the wild-type amyB gene locus relative to the total number of clones analyzed.

[0288] As depicted in FIG. 3 CRISPR/Cas9 based amyB gene deletion plasmids pCC010, pCC019 and pCCO22 are not functional in Bacillus licheniformis as all cells analyzed carried the wild-type amyB locus.

[0289] The other promoter variants are functional in Bacillus licheniformis driving the expression of Cas9. In particular, gene deletion plasmids pCC014, pCC016, pCCO25 with promoter variants PV4-5, PV4-7 and PV8-7 respectively show highest gene deletion efficiency with greater 60%.

[0290] A single correct clone was steaked onto fresh LB-agar plates without antibiotics following second incubation at 48° C. overnight for plasmid curing. Final clones were again analyzed for successful amyB gene deletion by colony PCR and plasmid loss analyzed by plating on LB-agar plates containing 20 μg/ml kanamycin. The resulting Bacillus licheniformis strain with cured deletion plasmid (sensitive to kanamycin) and deleted amyB gene was named Bacillus licheniformis P310.

Example 2: Gene Deletion and Gene Mutation with Promoters PV4-5 and PV8-7 in Bacillus licheniformis

[0291] Electrocompetent Bacillus licheniformis P308 cells were prepared as described above and transformed with 1 μg of each of the hag deletion plasmids pCCO29 and pCC030 with promoters PV4-5 (SEQ ID 037) PV8-7 (SEQ ID 046) respectively isolated from E.° coli INV110 cells (Life technologies) following plating on LB-agar plates containing 20 μg/ml kanamycin and incubation overnight at 37° C.

[0292] The next day 20 clones of each transformation reaction were subjected to colony-PCR to analyze for successful CRISPR/Cas9-based deletion of the hag gene and with oligonucleotides SEQ ID 087 and SEQ ID 088, and further transferred onto fresh LB-agar plates without antibiotics following incubation at 48° C. overnight for plasmid curing.

[0293] The efficiency of hag gene deletion for each CRISPR/Cas9-based deletion plasmid was calculated as the ratio in percentage of successful gene deletion based on the appearance of the expected smaller specific PCR-amplicon compared to the larger specific PCR-amplicon of the wild-type hag gene locus relative to the total number of clones analyzed.

[0294] The experiment for each hag gene deletion plasmid was performed three times. As depicted in FIG. 4A the CRISPR/Cas9-based hag gene deletion efficiencies of plasmids pCCO29 and pCC030 are 95% and 100% respectively.

[0295] To analyze the efficiency for introduction of point mutations, Bacillus licheniformis P308 cells were transformed with two degU mutation plasmids pCC031 and pCC032 as described for deletion of the hag gene, again differing in the promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046) driving the constitutive expression of Cas9. The transformed Bacillus licheniformis cells were plated on LB-agar plates containing 20 μg/ml kanamycin following incubation overnight at 30° C. The mutation efficiency of introduction of the H12L degU mutation was calculated as the ratio in percentage of successful mutated degU gene based on the appearance of a degU-specific PCR-amplicon with oligonucleotides SEQ ID 089 and SEQ ID 090 that can be cleaved with the restriction endonuclease PstI compared to the native degU-specific PCR-amplicon of the wild-type degU gene locus relative to the total number of 20 clones analyzed. The experiment for each degU mutation plasmid was performed three times. As depicted in FIG. 4B the CRISPR/Cas9-based mutation efficiencies of plasmids pCC031 and pCC032 are 19% and 24% respectively.

Example 3: Gene Deletion with Promoters PV4-5 and PV8-7 in Bacillus subtilis

[0296] Electrocompetent Bacillus subtilis ATCC6051a cells were prepared as described above and transformed with 1 μg of each of the amyE deletion plasmids pCC033 and pCC034 with promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046) respectively isolated from E.° coli DH10B cells following plating on LB-agar plates containing 20 μg/ml kanamycin and incubation overnight at 37° C.

[0297] The next day 20 clones of each transformation reaction were subjected to colony-PCR to analyze for successful CRISPR/Cas9-based deletion of the amyE gene with oligonucleotides SEQ ID 091 and SEQ ID 092, and further transferred onto fresh LB-agar plates without antibiotics following incubation at 48° C. overnight for plasmid curing.

[0298] The efficiency of amyE gene deletion for each CRISPR/Cas9-based deletion plasmid was calculated as the ratio in percentage of successful gene deletion based on the appearance of the expected smaller specific PCR-amplicon compared to the larger specific PCR-amplicon of the wild-type amyE gene locus relative to the total number of clones analyzed.

[0299] The experiment for each amyE gene deletion plasmid was performed three times. As depicted in FIG. 5A the CRISPR/Cas9-based amyE gene deletion efficiencies of plasmids pCC033 and pCC034 within Bacillus subtilis are 97% and 100% respectively.

[0300] The gene deletion efficiency of plasmids pCC035 and pCC036 in dependency of promotors PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046) for deletion of the aprE gene of Bacillus subtilis was analyzed similar to the procedure described for the deletion of the amyE gene, however cells were incubated on LB-agar plates containing 20 μg/ml kanamycin after transformation at 30° C. overnight. The gene deletion was again analyzed by colony-PCR with oligonucleotides SED ID 093 and SEQ ID 094 and the gene deletion efficiency calculated as described above for three independent transformation reactions. As depicted in FIG. 5B the CRISPR/Cas9-based aprE gene deletion efficiencies of plasmids pCC035 and pCC036 within Bacillus subtilis are 32% and 47% respectively.

Example 4: Gene Deletion with Promoters PV4-5 and PV8-7 and Different Spacers in Bacillus licheniformis

[0301] Electrocompetent Bacillus licheniformis Bli #005 cells were prepared as described above and transformed with 1 μg of each of the vpr deletion plasmids pCC037, pCC038 and pCC039 with promoter PV4-5 (SEQ ID 037) and different vpr-specific spacer sequences (SEQ ID 066-068) respectively isolated from E.° coli Ec #098 cells following plating on LB-agar plates containing 20 μg/ml kanamycin and incubation overnight at 37° C.

[0302] The next day 20 clones of each transformation reaction were subjected to colony-PCR to analyze for successful CRISPR/Cas9-based deletion of the vpr gene with oligonucleotides SEQ ID 095 and SEQ ID 096, and further transferred onto fresh LB-agar plates without antibiotics following incubation at 48° C. overnight for plasmid curing.

[0303] The efficiency of vpr gene deletion for each CRISPR/Cas9-based deletion plasmid was calculated as the ratio in percentage of successful gene deletion based on the appearance of the expected smaller specific PCR-amplicon compared to the larger specific PCR-amplicon of the wild-type vpr gene locus relative to the total number of clones analyzed. As depicted in FIG. 6A the CRISPR/Cas9-based vpr gene deletion efficiency of plasmids pCC037, pCC038 and pCC039 is 100%, 100% and 84% respectively.

[0304] The gene deletion efficiency of plasmids pCC040. pCC041 and pCC042 with promoter PV4-5 (SEQ ID 037) and different epr-specific spacer sequences (SEQ ID 070-072) for deletion of the epr gene of Bacillus licheniformis was done as described for the vpr gene, however, oligonucleotides SEQ ID 097 and SEQ ID098 were used for colony-PCR-based analysis of the gene deletion. As depicted in FIG. 6B the CRISPR/Cas9-based epr gene deletion efficiency of plasmids pCC040, pCC041 and pCC042 is 87.5%, 100% and 100% respectively.

Example 5: Gene Integration with Promoters PV4-5 and PV8-7 in Bacillus licheniformis

[0305] Electrocompetent Bacillus licheniformis Bli #005 cells were prepared as described above and transformed with 1 μg of the gene integration plasmid pCC043 with promoter PV4-5 (SEQ ID 037) isolated from E.° coli Ec #098 cells following plating on LB-agar plates containing 20 μg/ml kanamycin and incubation overnight at 37° C.

[0306] The next day 20 clones of the transformation reaction were subjected to colony-PCR with oligonucleotides SEQ ID 009 and SEQ ID 010 to analyze for successful CRISPR/Cas9-based integration of the PaprE-GFPmut2 expression cassette to replace the amyB gene of Bacillus licheniformis, and further transferred onto fresh LB-agar plates without antibiotics following incubation at 48° C. overnight for plasmid curing.

[0307] The efficiency of gene integration for the pCC043 CRISPR/Cas9-based gene integration plasmid was calculated as the ratio in percentage of successful gene integration based on the appearance of the expected specific PCR-amplicon compared to the larger specific PCR-amplicon of the wild-type amyB gene locus relative to the total number of clones analyzed.

[0308] The experiment was performed twice. As depicted in FIG. 7 the CRISPR/Cas9-based gene integration efficiency of plasmid pCC043 into Bli #005 is 67%.

[0309] The efficiency of the gene integration of the PaprE-GFPmut2 expression cassette with plasmid pCC043 was similarly determined for the Bacillus licheniformis P308 strain showing in two independent transformation reactions an average gene integration efficiency of 72% as depicted in FIG. 7.

Example 6: Gene Deletion with Promoters PV4-5 in Bacillus pumilus

[0310] Electrocompetent Bacillus pumilus DSM14395 cells were prepared as described above and transformed with 1 μg each of the sporulation gene deletion plasmids pCC044 (sigE), pCC045 (sigF) and pCC046 (spoIIE) with promoter PV4-5 (SEQ ID 037) driving the expression of the Cas9 endonuclease. The plasmid DNA was isolated from E.° coli DH10B cells and in vitro methylated as described above prior to transformation. Transformed Bacillus pumilus cells were plated on LB-agar plates containing 20 μg/ml kanamycin and incubated overnight at 37° C.

[0311] The next day 20 clones of each of the transformation reactions were subjected to colony-PCR with oligonucleotides SEQ ID 099 and SEQ ID 100 for analysis of the sigE deletion, with oligonucleotides SEQ ID 101 and SEQ ID 102 for analysis of the sigF deletion and with oligonucleotides SEQ ID 103 and SEQ ID 104 for analysis of the spoIIE deletion. Individual colonies were further transferred onto fresh LB-agar plates without antibiotics following incubation at 48° C. overnight for plasmid curing.

[0312] The efficiencies of the gene deletion of plasmids pCC044, pCC045 and pCC046 in Bacillus pumilus were calculated as the ratio in percentage of successful gene deletion based on the appearance of the expected smaller specific PCR-amplicon compared to the larger specific PCR-amplicon of the wild-type gene locus relative to the total number of clones analyzed.

[0313] As depicted in FIG. 8 the CRISPR/Cas9-based gene deletion efficiencies of plasmids pCC044, pCC045 and pCC046 within Bacillus pumilus are 43%, 56% and 50% respectively.