A shuttle vector for expression in Escherichia coli and Bacillus

Abstract

The present invention belongs to the field of molecular biology and provides a shuttle vector for expression in Escherichia coli (E. coli) and Bacilli, said shuttle vector comprising 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 that confers reduced constitutive expression compared to the corresponding initiation regulatory nucleic acid molecule in bacterial cells.

Description

SUMMARY OF THE INVENTION

[0002] The present invention belongs to the field of molecular biology and provides a shuttle vector for expression in Escherichia coli (E. coli) and Bacilli, said shuttle vector comprising 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 that confers reduced constitutive expression compared to the corresponding initiation regulatory nucleic acid molecule in a bacterial cell.

[0003] INTRODUCTION TO THE INVENTION

[0004] Microorganisms are widely used in industry today by harnessing their fermentation capabilities. Microorganisms are particularly used as hosts for the fermentative production of a variety of substances such as enzymes, proteins, chemicals, sugars, and polymers. For these purposes, microorganisms are the subject of genetic engineering aimed at modifying their gene expression to meet the requirements of specific production processes. Rational genetic engineering of microorganisms requires target-specific genome editing techniques such as the introduction of point mutations, gene deletions, gene insertions, and gene duplications.

[0005] Many different genome editing methods have been developed for several species. Most of them require the introduction of double-stranded DNA breaks or two adjacent single-stranded DNA breaks to introduce random mutations at specific loci in the genome by non-homologous end joining (NHEJ) or to introduce, replace, or delete DNA using a homologous recombination repair mechanism (HR) that requires the delivery of a donor DNA molecule. Techniques used are, for example, zinc finger nucleases, TALENs, homing endonucleases, etc. The recent development of CRISPR (clustered regularly interspaced short palindromic repeats)-based systems has made genome editing even more attractive due to its precision, efficiency, and speed.

[0006] The CRISPR system was initially identified as an adaptive defense mechanism in bacteria of the genus Streptococcus (WO2007 / 025097). Those bacterial CRISPR systems rely on a guide RNA (gRNA) complexed with a nuclease protein to direct the degradation of complementary sequences present within the invasive viral DNA. The first identified protein in the CRISPR / Cas system, Cas9, is a large monomeric DNA nuclease that is directed by a complex of two non-coding RNAs (crRNA and trans-activating crRNA (tracrRNA)) to a DNA target sequence adjacent to a PAM (protospacer adjacent motif) sequence motif. Subsequently, it was shown that a synthetic RNA chimera (single guide RNA or sgRNA) created by fusing crRNA with tracrRNA is equally functional (Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. and Charpentier, E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096), 816-821. 17-8-2012).

[0007] Several research groups have found that the CRISPR cleavage property can be used to disrupt genes in the genomes of almost any organism with unprecedented ease (Mali P et al. (2013) Science. 339(6121):819-823; Cong L et al. (2013) Science 339(6121)). Recently, it has become clear that providing a template for repair allows editing of the genome at almost any site with almost any desired sequence, thus transforming CRISPR into a powerful gene editing tool (WO / 2014 / 150624, WO / 2014 / 204728).

[0008] The key element driving gene expression in host cells is the promoter sequence. For gene expression to occur, RNA polymerase must bind to the promoter sequence near the gene. Thus, a promoter contains specific DNA sequences that provide binding sites for RNA polymerase and also for other proteins (i.e., transcription factors) that recruit RNA polymerase to the recognition sequence. In bacteria, promoters are typically recognized by RNA polymerase and associated σ factors, which are directed to the promoter DNA by activator proteins binding to their own DNA binding sites nearby (Lee, D.J., Minchin, S.D. and Busby, S.J. Activating transcription in bacteria. Annu. Rev. Microbiol. 66, 125 - 152. 2012). For example, constitutive promoters that drive the expression of many housekeeping genes are independent of activation or derepression by activator or repressor proteins and RNA polymerase binds to the constitutive promoter through the associated σ factor sigA (also called sig70 in Escherichia coli) that recognizes the sigA - specific DNA sequence elements, the - 35 box and the - 10 box. SigA - dependent promoters in Bacillus and Escherichia coli have been well - studied and comparison of the consensus motifs of sigA promoter sequences suggests that Bacillus - derived and Escherichia coli - derived sigA promoters are cross - recognized by Escherichia coli RNA polymerase and Bacillus RNA polymerase and the corresponding sig70 and sigA factors respectively (Helmann, J.D. Compilation and analysis of Bacillus subtilis σA - dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res. 23(13), 2351 - 2360. 11 - 7 - 1995).

[0009] In eukaryotes, the process is more complex and multiple factors are required for RNA polymerase to bind to the promoter. Influenced by nucleic acid sequences, promoters can confer low, medium or high expression levels and can be constitutive or inducible.

[0010] Numerous constitutive promoters of Bacillus have been described. The promoter Pveg of the veg gene is a well-described constitutive strong promoter. Additionally, a library of expression modules containing constitutive promoters of Bacillus with different promoter strengths has been constructed (Guiziou, S., et al. (2016). Nucleic Acids Res. 44(15), 7495-7508).

[0011] By adding an inducer molecule to the cell, an inducible promoter is activated or derepressed. Thus, an activator protein binds to a sequence adjacent to the promoter sequence and actively recruits RNA polymerase and associated σ factors to allow transcription initiation. Well-known examples described are the E. coli P promoter regulated by araC which changes the promoter conformation upon addition of arabinose and binds as a dimer to the operator sites I1 and I2 BAD promoter and the Bacillus mannitol-inducible promoter system PmanP regulated by the activator manR. Inducible promoters such as the lacUV5 promoter, the T7-phage promoter for expression in E. coli, and the Bacillus Pspac-I and Ppac-I promoters are negatively regulated by the lac repressor (encoded by the lacI gene), which binds to its specific lac operator site either within (e.g., between the -35sigA recognition site and the -10sigA recognition site) or near (i.e., 3’ or 5’ of the promoter sequence) the promoter sequence in the absence of an inducer molecule to prevent transcription. Another example is the PxylA inducible promoter system from Bacillus megaterium which is widely used in Bacillus expression systems. The PxylA promoter is negatively regulated by the xylR repressor protein containing the xylR operator site at the 3’ of the transcription start site.

[0012] Inducible promoter systems generally facilitate the cloning process in expression vectors because gene expression under the control of such promoters is greatly reduced and thus, for example, the adverse effects regarding depletion of cellular resources, interference with cell metabolism, etc. are minimized. However, careful analysis of the adjustment of the desired protein expression with respect to the amount of inducer molecule added and the time point of induced expression is required for each strain in which the promoter is used. In contrast, constitutive promoters have the advantage of being independent of the application of an inducer, do not require specific regulators or transporter proteins, and are thus active in a wide variety of bacteria.

[0013] A plasmid is an extrachromosomal circular DNA that replicates autonomously in a host cell and is thus independent of host chromosome replication.

[0014] For autonomous replication, plasmids contain an origin of replication that enables the vector to replicate autonomously in the host cell under discussion. Examples of bacterial origins of replication are the origins of replication of plasmids pUB110, pE194, pC194, pTB19, pAMβ1, pTA1060, which allow replication in Bacillus, and the origins of replication of plasmids pBR322, colE1, pUC19, pSC101, pACYC177, and pACYC184, which allow replication in Escherichia coli (Sambrook, J. and Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001).

[0015] The plasmid copy number is defined as the average number of plasmid per bacterial cell or per chromosome under normal growth conditions. Additionally, there are different types of origins of replication (also called replicons) that result in different copy numbers in bacterial hosts.

[0016] Plasmid replicons pBS72 and plasmids pTB19 and derivatives pTB51, pTB52 confer low copy numbers, 6 copies and 1 - 8 copies respectively in Bacillus cells, while plasmids pE194 and pUB110 confer low - medium copy numbers, 14 - 20 copies per cell respectively, and medium copy numbers, 30 - 50 copies per cell respectively. Plasmid pE194 has been analyzed in more detail (Villafane, et al. (1987): J. Bacteriol. 169(10), 4822 - 4829) and several pE194–cop mutants with high copy numbers ranging from 85 copies to 202 copies inside Bacillus have been described. Additionally, plasmid pE194 is temperature - sensitive, with a stable copy number up to 37 °C, however replication is eliminated above 43 °C. Additionally, there is a variant of pE194 called pE194ts, with 2 point mutations inside the replicon region, resulting in more exaggerated temperature - sensitivity - stable copy number up to 32 °C, however only 1 - 2 copies per cell at 37 °C.

[0017] In Escherichia coli, the pBR322 plasmid carrying the pMB1 replicon or its close relative, the colicin E1 (colE1) replicon, maintains a low-to-medium copy number, i.e., 15-20 copies per bacterial cell. Deletion of the rop / rom gene within the colE1 and pMB1 plasmid derivatives slightly increases the plasmid copy number inside E. coli cells to a medium copy number of 25-50. The pUC vector series are small high-copy plasmids with up to 200 copies per E. coli cell, derived from a mutant pBR322 plasmid lacking the rop protein. The pUC plasmids are well-established cloning vectors due to their small size and high yield during plasmid preparation compared to the pBR322-derived and ColE1-derived vectors mentioned above.

[0018] Alternatively, the p15A replicon present in the pACYC177 / 184 plasmids confers a low-to-medium copy number of 20 copies per cell, and the pSC101 replicon confers a low copy number of 5-10 copies per cell. Plasmids with low to medium copy numbers and encoding toxic or unfavorable expression constructs are generally stably maintained inside cells; however, the yield during plasmid preparation is low. For subsequent transformation of bacterial cells, the amount of plasmid DNA becomes limiting compared to plasmid preparation of high-copy plasmids. This is especially relevant for medium- to high-throughput applications when multiple preparations are carried out in parallel.

[0019] The combination of plasmid copy number and promoter selection for gene expression determines the overall protein expression level and thus affects cell viability and plasmid stability.

[0020] CRISPR-based expression systems applied to Gram-positive organisms (such as Bacillus species) have been successfully applied. The expression systems are based on a single plasmid system approach, i.e., containing the Cas9 endonuclease, gRNA (such as sgRNA or crRNA / tracrRNA), and repair homology sequences (donor DNA) on a single Escherichia coli - Bacillus shuttle vector.

[0021] Altenbuchner created a series of high-copy pUC replicons, which are Escherichia coli-Bacillus subtilis shuttle plasmids for B. subtilis based on CRISPR / Cas9 genome editing, in combination with the inducible promoters PmanP, PxylA, and PtetLM to express the Cas9 endonuclease (Altenbuchner, (2016): Applied and environmental microbiology 82(17), 5421-5427). This allows for highly efficient plasmid DNA preparation and stable maintenance inside the E. coli cloning host. Similarly, a similar method was developed to construct a high-copy pUC-derived CRISPRi-Escherichia coli-Bacillus subtilis shuttle plasmid for use in Bacillus methanolicus. The promoter of the mannitol-activating gene mtlR of B. methanolicus that drives the expression of defective Cas9 was modified by introducing a lacO site at the 3' of the promoter, thus effectively blocking transcriptional activity with intact lacI in E. coli( et al. (2019): Applied microbiology and biotechnology 103(14), 5879–5889).

[0022] Another single plasmid scheme for the application of CRISPR / Cas9 in B. subtilis uses the low to medium copy number replicon p15A in combination with an inducer-independent promoter (B. amyloliquefaciens PamyQ-amylase promoter) that expresses Cas9, allowing for the successful cloning and stable maintenance of an Escherichia coli-Bacillus subtilis shuttle plasmid for CRISPR / Cas9-based genome editing in E. coli. A similar combination of a medium copy pBR322-derived Escherichia coli-Bacillus subtilis shuttle vector and Cas9 under the control of a constitutive strong promoter was applied (Zhou et al. (2019): International journal of biological macromolecules 122, 329–337).

[0023] Although low-copy and medium-copy backbones reduce the metabolic burden, this is accompanied by a reduced plasmid yield from Escherichia coli and impedes the isolation of plasmid DNA on the scale required by many transformation protocols that are difficult to transform Bacillus strains or difficult to apply in high-throughput applications. Inducer-dependent promoter systems are not always applicable in a wide variety of different microorganisms and furthermore require the analysis of the amount of inducer-molecule and the time point for inducing the promoter. Additionally, compared to constitutive promoters, an additional promoter activation step by adding inducer molecules to the cells is required, which lengthens the overall time frame of the genome editing method.

[0024] Accordingly, there is a need in the art to provide vectors and systems that allow the use of high-copy vectors in combination with the use of constitutive promoters to overcome these limitations. DETAILED DESCRIPTION OF THE INVENTION

[0026] A first embodiment of the present invention is a shuttle vector comprising a high-copy origin of replication (ORI) functional in Escherichia coli and a low-to-medium-copy ORI functional in Bacillus, and a synthetic constitutive regulatory nucleic acid that confers reduced constitutive expression compared to the corresponding initiation regulatory nucleic acid molecule in a bacterial cell.

[0027] Another embodiment of the present invention is the shuttle vector, wherein the synthetic constitutive regulatory nucleic acid is operably linked to a coding region that, upon high expression, will stress the bacterium, thereby resulting in a reduced growth rate or growth potential of the bacterium, preferably a coding region encoding a TALEN, a homing endonuclease, a meganuclease, or a CRISPR / Cas enzyme, preferably a cas9 or Cas12a enzyme.

[0028] Reduced growth means that after incubating for a certain period of time under conditions suitable for the corresponding bacterium on a plate, there is a visible difference in the visible size of the corresponding colonies between the bacterial colonies containing the construct as described above and the bacterial colonies not containing the construct. For example, the bacterial colonies containing the construct will show smaller colonies compared to the bacterial colonies not containing the construct. For example, Escherichia coli bacteria will be incubated at 36 - 37 °C for 8 - 16 hours, and then the difference in colony size will be compared.

[0029] The coding region that subjects the bacterium expressing the coding region under the control of a constitutive strong promoter can be, for example, any coding region encoding a protein greater than 150 kDa (such as Cas9 or Cas12a), a coding region encoding an enzyme that induces DNA strand breaks or mutations (such as Cas9, Cas12a, and any other CRISPR Cas enzyme, homing endonuclease, meganuclease, adenosine deaminase, or DNA glycosylase), a coding region encoding an enzyme that interferes with bacterial metabolism (such as an enzyme involved in generating energy equivalents (ATP) or cofactors such as NADP), or a coding region encoding a transporter or transmembrane protein that interferes with substrate uptake or bacterial cell detoxification.

[0030] Constitutive expression in a bacterial cell means that the expression intensity derived from the corresponding promoter is substantially constant under various conditions. In this specification, constitutive expression means that the expression derived from a promoter varies by less than 10-fold, preferably less than 9-fold, preferably less than 8-fold, preferably less than 7-fold, preferably less than 6-fold, preferably less than 5-fold, preferably less than 4-fold, more preferably less than 3-fold, even more preferably less than 2-fold under the following conditions: in rich medium, such as LB medium, in rich medium substituted with sugar, such as sucrose, lactose, or glucose, preferably at a concentration between 0.1% and 0.5%, preferably 0.3% glucose, and in minimal salt medium, such as M9 medium supplemented with sugar, such as sucrose, lactose, or glucose, preferably at a concentration between 0.1% and 0.5%, preferably 0.3% glucose, during the exponential growth phase, transition phase, and stationary phase.

[0031] To determine whether a gene is differentially expressed, gene expression is measured at least in triplicate across these conditions and the difference in these values is measured using the standard method in the art, the DESeq2 software package (Love, M.I. et al., Genome Biology 15(12):550 (2014)). This analysis will evaluate the observed fold change between conditions and the probability that this difference is due to random chance. Any gene that is more upregulated or downregulated than defined above and has a probability of less than 5% due to random chance is considered differentially expressed and thus not constitutively expressed.

[0032] A constitutive promoter is independent of other cellular regulators and transcriptional initiation depends on sigma factor A (sigA). The sigA-dependent promoter contains the sigma factor A-specific recognition sites, the '-35'-region and the '-10'-region.

[0033] Preferably, the constitutive promoter sequence is selected from the group consisting of: promoters Pveg, PlepA, PserA, PymdA, Pfba with different gene expression intensities and their derivatives (Guiziou et al., (2016): Nucleic Acids Res. 44(15), 7495-7508), phage SPO1 promoters P4, P5, P15 (WO15118126), the cryIIIA promoter from Bacillus thuringiensis (WO9425612), and combinations thereof, or active fragments or variants thereof.

[0034] A replication origin (ORI) conferring a high copy number means an ORI that results in at least 51 copies of the corresponding vector in the corresponding bacterial cell in which the ORI is functional. Since the copy number depends on the temperature at which the corresponding bacteria are cultured, preferably this definition refers to the temperature at which the corresponding bacteria are cultured in a laboratory known to the person skilled in the art, such as described for various strains (Bronikowski et al. (2001): Evolution 55(1): 33-40).

[0035] Preferably for Escherichia coli, this means detecting the copy number during growth at 36-37 °C, and for Bacillus, this means detecting the copy number during growth at 36-37 °C.

[0036] An ORI conferring a medium copy number means an ORI that maintains 25-50 vector copies, an ORI conferring a low-medium copy number means an ORI that maintains 11-24 copies per cell, and an ORI conferring a low copy number means an ORI that maintains 1-10 vector copies inside the bacterial cell.

[0037] In a preferred embodiment, the Escherichia coli ORI is selected from high copy number ORIs and the Bacillus ORI is selected from low copy number ORIs, low-medium copy number ORIs, and medium copy number ORIs.

[0038] More preferably, the Escherichia coli ORI is selected from high copy number ORIs and the Bacillus ORI is selected from low-medium copy number ORIs.

[0039] More preferably, the Escherichia coli ORI is selected from high copy number ORIs and the Bacillus ORI is selected from temperature-sensitive low-medium copy number ORIs, such as plasmid pE194 derivatives that confer low-medium copy numbers at 36-37 °C and low-medium copy numbers at 30-33 °C and do not replicate above 43 °C.

[0040] More preferably, the E. coli ORI is selected from high-copy number ORIs, such as, for example, pUC ORI, and the Bacillus ORI is selected from temperature-sensitive low-medium copy number ORIs such as plasmid pE194ts derivatives that confer low copy number at 36-37 °C, low-medium copy number at 30-33 °C and do not replicate above 38 °C.

[0041] The term "clone showing a growth rate comparable to that of the corresponding WT strain not containing the construct" means a clone transformed with the construct as defined above, which shows a growth rate of at least 50% of the growth rate of the WT bacteria when compared to bacteria not containing such a construct or not transformed with it. Preferably they have a growth rate of at least 60%, 65%, 70%, 75%, 80%, 85% of the WT bacteria. More preferably, they have a growth rate of at least 90%, 95% of the WT bacteria or have the same growth rate as the WT bacteria. The growth rate can be determined, for example, based on the cell density after a certain incubation time in liquid culture or based on the colony size on a plate.

[0042] Functional expression of a coding region means that the expression of such a coding region is detectable at least, for example, by RNA detection methods such as RT-PCR, qPCR or by gene deletion efficiency using a detectable protein such as a fluorescent protein, GUS, an enzyme reaction specific for the corresponding enzyme or a coding region encoding an enzyme that induces double-strand breaks in the genome (such as a CRISPR / Cas enzyme).

[0043] Another embodiment of the invention is a shuttle vector as defined above, wherein the starting regulatory nucleic acid molecule conferring constitutive expression in bacterial cells is selected from

[0044] a) SEQ ID NO:28 and 29,

[0045] b) a nucleic acid molecule comprising at least 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs identical to 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs of the sequence as set forth in SEQ ID NO:28 or 29, and

[0046] c) a nucleic acid molecule having at least 90% identity, preferably at least 91%, 92%, 93%, 94% or 95%, more preferably at least 96%, 97%, 98% or 99% identity over the entire length of the sequence as set forth in SEQ ID NO:28 or 29, and

[0047] d) A nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule of at least 20 consecutive base pairs, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs of the nucleic acid molecule of SEQ ID NO: 28 or 29,

[0048] e) The complementary sequence of any one of the nucleic acid molecules as defined in a) to d).

[0049] Another embodiment of the present invention is a shuttle vector as defined above, wherein the synthetic regulatory nucleic acid molecule is included in the group consisting of

[0050] A) A nucleic acid molecule having a sequence of SEQ ID NO 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

[0051] B) A nucleic acid molecule which comprises at least 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs identical to 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs of the sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

[0052] C) A nucleic acid molecule which has at least 90% identity, preferably at least 91%, 92%, 93%, 94% or 95%, more preferably at least 96%, 97%, 98% or 99% identity over its entire length to the sequence of SEQ ID NO: 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

[0053] D) A nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule of at least 20, preferably 25, more preferably 50, more preferably 75, more preferably 100, even more preferably 110, even more preferably 120 consecutive base pairs of any one of the nucleic acid molecules of SEQ ID NO: 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and

[0054] E) The complementary sequence of any one of the nucleic acid molecules as defined in A) to D),

[0055] wherein the sequences as defined in B) to E) are different from the corresponding starting regulatory nucleic acid molecule having SEQ ID NO 28 or 29 and preferably contain at least one base deletion or insertion compared to the corresponding starting regulatory nucleic acid.

[0056] In yet another embodiment of the invention, the shuttle vector of the invention comprises a synthetic regulatory nucleic acid molecule as described above, wherein a method is applied to generate the nucleic acid molecule, the method comprising the steps

[0057] a. identifying at least one initial regulatory nucleic acid molecule that confers constitutive expression in bacterial cells, and

[0058] b. operably linking the initial regulatory nucleic acid molecule to a coding region encoding a protein heterologous to the initial regulatory nucleic acid molecule, and

[0059] c. introducing into a vector a construct comprising the initial regulatory nucleic acid molecule operably linked to the coding region, the vector comprising an origin of replication that confers a high copy number of the vector in bacterial cells, wherein the construct confers high expression of the coding region, and wherein the high expression of the coding region in bacterial cells subjects the bacterial cells to stress, resulting in reduced growth or elimination, and

[0060] d. transforming the vector into bacterial cells, and

[0061] e. culturing the transformed bacterial cells to recover a single clone, and

[0062] f. isolating a single clone that exhibits a growth rate comparable to that of a corresponding bacterial strain not containing the construct, and

[0063] g. isolating the construct from the clone; and

[0064] h. testing the functional expression of a gene operably linked to the synthetic regulatory nucleic acid molecule contained in the construct and optionally

[0065] i. sequencing the corresponding regulatory nucleic acid molecule contained in the construct, thereby identifying a synthetic regulatory nucleic acid molecule that confers reduced constitutive expression in bacterial cells.

[0066] In yet another embodiment of the invention, the shuttle vector of the invention comprises a pUC ORI for replication in Escherichia coli (E. coli), a pE194ts ORI for replication in Bacillus spp., a selectable marker, and a synthetic regulatory nucleic acid molecule as defined above.

[0067] Preferably, the shuttle vector of the present invention comprises a pUC ORI for replication in Escherichia coli (E. coli), a pE194ts ORI for replication in Bacillus spp., a selectable marker, and a synthetic regulatory nucleic acid molecule selected from SEQ ID NO: 37, 39, 46 and functional derivatives thereof as defined above under B) to E).

[0068] In a preferred embodiment, the synthetic regulatory nucleic acid molecule comprised in the shuttle vector of the present invention is functionally linked to a coding region encoding a TALEN, a homing endonuclease, a meganuclease, a zinc finger protein or a CRISPR / Cas protein, preferably a coding region encoding a CRISPR / Cas protein, more preferably a coding region encoding a Cas9 or Cas12a protein.

[0069] In yet another embodiment, a shuttle vector comprising a pUC ORI for replication in Escherichia coli, a pE194ts ORI for replication in Bacillus spp., a selectable marker, and a synthetic regulatory nucleic acid molecule driving the expression of a CRISPR / Cas endonuclease further comprises a constitutive promoter driving the expression of spacer-sgRNA.

[0070] In yet another embodiment, a shuttle vector comprising a pUC ORI for replication in Escherichia coli, a pE194ts ORI for replication in Bacillus spp., a selectable marker, a synthetic regulatory nucleic acid molecule driving the expression of a CRISPR / Cas endonuclease, and driving the expression of spacer-sgRNA further comprises a donor DNA molecule.

[0071] Yet another embodiment of the present invention is a method for expressing a coding region in a bacterium, wherein when highly expressed, the coding region will stress the bacterium, resulting in a decrease in the growth rate or growth potential of the bacterium, the method comprising introducing into the bacterium the shuttle vector of the present invention, wherein the coding region is functionally linked to the synthetic constitutive regulatory nucleic acid conferring reduced constitutive expression. Preferably, wherein the coding region is a protein essential for genome editing. More preferably, the coding region encodes a TALEN, a homing endonuclease, a meganuclease or a CRISPR / Cas enzyme, most preferably a cas9 or Cas12a enzyme.

[0072] In yet another embodiment of the method of the present invention, the bacterium is a Gram-positive or Gram-negative bacterium, preferably belonging to the class Bacilli or Gammaproteobacteria; more preferably, belonging to the family Bacillaceae or Enterobacteriaceae; even more preferably, belonging to the genus Bacillus or Escherichia; even more preferably, belonging to the genus Bacillus.

[0073] Preferred Bacillus bacteria include 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 cells.

[0074] Preferably, the bacterium comprises at least three different Bacillus species, at least two different Bacillus species or at least one Bacillus species.

[0075] More preferably, the Bacillus species includes at least one of the following: Bacillus subtilis, Bacillus licheniformis or Bacillus pumilus. Most preferably, the bacterium is Bacillus licheniformis.

[0076] Another embodiment of the present invention is a system for expressing a coding region encoding a protein, the expression of which will stress the bacteria, the system comprising the shuttle vector of the present invention and a coding region heterologous to the constitutive regulatory nucleic acid, which confers reduced constitutive expression compared to the corresponding starting regulatory nucleic acid molecule in a bacterial cell. In a preferred embodiment of the system of the present invention, the coding region always encodes a protein essential for genome editing, preferably a TALEN, a homing endonuclease, a meganuclease or a CRISPR / Cas enzyme, more preferably a Cas9 or Cas12a enzyme.

[0077] Definitions

[0078] Abbreviations: GFP – green fluorescent protein, GUS – β-galactosidase, BAP – 6-benzylaminopurine, 2,4-D – 2,4-dichlorophenoxyacetic acid, MS – Murashige-Skoog medium, NAA – 1-naphthylacetic acid, MES, 2-(N-morpholino)-ethanesulfonic acid, IAA: indoleacetic acid, Kan: kanamycin sulfate, GA3 – gibberellic acid, Timentin TM : ticarcillin disodium / clavulanate potassium, microl: microliter.

[0079] It should be understood that the present invention is not limited to specific methods or protocols. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, etc. The term "about" is used herein to mean approximately, roughly, around and within the range of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundary values above and below the numerical value. Generally, the term "about" is used herein to modify a numerical value above and below the stated value by a variation of 20%, preferably 10% above or below (higher or lower). As used herein, the word "or" means any one of the specifically listed members and also includes any combination of the listed members. When used in this specification and the following claims, the words "comprise", "comprising", "include", "including" and "includes" are intended to specify the presence of one or more of the stated features, integers, components or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps or groups thereof. For clarity, certain terms used in this specification are defined and used as follows.

[0080] Coding region: As used herein, the term "coding region", when referring to the use of a structural gene, refers to the nucleotide sequence that encodes the amino acids present in the nascent polypeptide that results from the translation of an mRNA molecule. The coding region is bounded on the 5' side by the nucleotide triplet "ATG" that encodes the initiator methionine and on the 3' side by three triplets (i.e., TAA, TAG, TGA) that specify a stop codon. Alternatively, the nucleotide triplet can be "GTG" or "TTG" and is considered an initiation nucleotide triplet because the ribosome binding site (Shine Dalgarno) is present 4 to 12 nucleotides 5' relative to the nucleotide triplet. The genomic form of a gene can also contain sequences located on the 5'-end and 3'-end of the sequence present in the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are 5' or 3' of the untranslated sequences present on the mRNA transcript). The 5'-flanking region can contain regulatory sequences such as promoters and enhancers that control or affect gene transcription and the ribosome binding site (Shine Dalgarno) that controls or affects mRNA translation. The 3'-flanking region can contain sequences that direct transcription termination and post-transcriptional cleavage.

[0081] Complementary: "Complementary" or "complementarity" refers to two nucleotide sequences that contain anti-parallel nucleotide sequences, where the anti-parallel nucleotide sequences are capable of pairing with each other (by the base-pairing principle) once hydrogen bonds are formed between complementary base residues in the anti-parallel nucleotide sequences. For example, the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity can be "partial" or "complete". "Partial" complementarity is a situation where one or more nucleic acid bases do not match according to the base-pairing rules. "Total" or "complete" complementarity between nucleic acid molecules is a situation where each nucleic acid base matches another base according to the base-pairing rules. The degree of complementarity between nucleic acid molecule strands has a significant effect on the efficiency and strength of hybridization between nucleic acid molecule strands. As used herein, the "complement" of a nucleic acid sequence refers to a nucleotide sequence whose nucleic acid molecule exhibits complete complementarity to the nucleic acid molecule of that nucleic acid sequence.

[0082] Donor DNA molecule: As used herein, the terms "donor DNA molecule", "repair DNA molecule", or "template DNA molecule", which are used interchangeably herein, mean a DNA molecule having a sequence to be introduced into the genome of a cell. It may be flanked at the 5' and / or 3' ends by sequences homologous or identical to sequences in the target region of the genome of said cell. It may contain sequences not naturally present in the corresponding cell such as an ORF, non-coding RNA, or regulatory element that is to be introduced into the target region or it may contain sequences homologous to the target region except for at least one mutation (gene editing): the sequence of the donor DNA molecule may be added to the genome or it may replace a sequence of the length of the donor DNA sequence in the genome.

[0083] Double-stranded RNA: A "double-stranded RNA molecule" or "dsRNA" molecule comprises a sense RNA fragment of a nucleotide sequence and an antisense RNA fragment of the nucleotide sequence, both of which comprise nucleotide sequences that are complementary to each other, thereby allowing the sense RNA fragment and the antisense RNA fragment to pair and form a double-stranded RNA molecule.

[0084] Endogenous: An "endogenous" nucleotide sequence refers to a nucleotide sequence present in the genome of an untransformed cell.

[0085] Expression: "Expression" refers to the biosynthesis of a gene product, preferably to the transcription and / or translation of a nucleotide sequence, such as an endogenous gene or a heterologous gene, in a cell. For example, in the case of a structural gene, expression involves the transcription of the structural gene into mRNA and optionally subsequent translation of the mRNA into one or more polypeptides. In other cases, expression may refer only to the transcription of DNA carrying an RNA molecule.

[0086] Expression construct: As used herein, an "expression construct" means a DNA sequence capable of directing the expression of a particular nucleotide sequence in a suitable plant part or plant cell, the DNA sequence comprising a promoter functional in the plant part or plant cell into which the DNA sequence is to be introduced, the promoter being operably linked to a nucleotide sequence of interest optionally operably linked to a termination signal. If translation is required, the DNA sequence generally also contains sequences necessary for the correct translation of the nucleotide sequence. The coding region may encode a protein of interest, but may also encode a functional RNA of interest in the sense or antisense orientation, such as RNAa, siRNA, snoRNA, snRNA, microRNA, ta-siRNA or any other non-coding regulatory RNA. An expression construct containing a nucleotide sequence of interest may be chimeric, meaning that one or more of the components of the expression construct are heterologous with respect to one or more of the other components of the expression construct. The expression construct may also be an expression construct that occurs naturally but has been obtained in a recombinant form for heterologous expression. However, in general, the expression construct is heterologous with respect to the host, i.e., the particular DNA sequence of the expression construct does not naturally occur in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression construct cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. With respect to cell development, the promoter may also be specific for a particular developmental stage (e.g., biofilm formation, sporulation).

[0087] Exogenous: The term "exogenous" refers to any nucleic acid molecule (e.g., a gene sequence) that has been introduced into the genome of a cell by experimental manipulation and may contain a sequence that is present in the cell, provided that the introduced sequence contains some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) and is thus different from the naturally occurring sequence.

[0088] Functional linkage: The term “functionally linked” or “functionally linked to” is understood to mean, for example, that a regulatory element (such as a promoter) and a nucleic acid sequence to be expressed and, if desired, other regulatory elements are arranged in such a way in succession that each regulatory element can perform its intended function to allow, modify, promote or influence the expression of the nucleic acid sequence. As a synonym, “operably linked” or “operably linked to” can be used. Depending on the arrangement of the nucleic acid sequence, the expression can result in sense or antisense RNA. For this purpose, a direct linkage in the chemical sense does not necessarily have to be required. Genetic control sequences such as enhancer sequences can also exert their action on the target sequence from a distant position or even from other DNA molecules. A preferred arrangement is an arrangement in which the nucleic acid sequence to be expressed is recombinantly located behind the sequence acting as a promoter, so that the two sequences are covalently linked to each other. The distance between the promoter sequence and the nucleic acid sequence to be recombinantly expressed is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, and very particularly preferably less than 50 base pairs. In a preferred embodiment, the nucleic acid sequence to be transcribed is located behind the promoter in such a way that the transcription start point is identical to the desired start of the chimeric RNA of the present invention. Functional linkages and expression constructs can be generated by means of conventional recombinant and cloning techniques as described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience; Gelvin et al. (eds.) (1990) Plant Molecular Biology Manual; Kluwer Academic Publisher, Dordrecht, The Netherlands). However, other sequences, such as linkers serving as specific cleavage sites for restriction enzymes or sequences serving as signal peptides, can also be located between the two sequences. The insertion of sequences can also result in the expression of fusion proteins.Preferably, an expression construct consisting of the ligation of a regulatory region such as a promoter and a nucleic acid sequence to be expressed may exist in the form of vector integration and be inserted into the plant genome, for example by means of a transformation method.

[0089] Gene: The term "gene" refers to a region that is operably linked to a suitable regulatory sequence capable of regulating the expression of a gene product (e.g., a polypeptide or a functional RNA) in some way. A gene includes untranslated regulatory regions (e.g., promoters, enhancers, repressors, etc.) located before (upstream) and after (downstream) the coding region (open reading frame, ORF) in DNA, and, as needed, intervening sequences (e.g., introns) between individual coding regions (e.g., exons). The term "structural gene" as used herein is intended to refer to a DNA sequence that is transcribed into mRNA, which is then translated into an amino acid sequence characteristic of a specific polypeptide.

[0090] "Gene editing" as used herein means introducing a specific mutation at a specific location in the genome of a cell. More advanced techniques can be applied, such as using the CRISPR Cas system and donor DNA or the CRISPR Cas system associated with mutagenic activity such as deaminase, to introduce gene editing through a precise editing process (WO15133554, WO17070632).

[0091] Genome and genomic DNA: The term "genome" or "genomic DNA" refers to the heritable information of a host organism. In eukaryotes, the genomic DNA includes the DNA of the nucleus (also referred to as chromosomal DNA), and also includes the DNA of plastids (e.g., chloroplasts) and other organelles (e.g., mitochondria). Preferably, the term "genome" or genomic "DNA" refers to the chromosomal DNA of the nucleus. In prokaryotes, the genomic DNA includes the chromosomal DNA inside the bacterial cell.

[0092] Heterologous: In the context of a nucleic acid molecule or DNA, the term "heterologous" refers to a nucleic acid molecule that is operably linked to or manipulated to become operably linked to a second nucleic acid molecule that is not operably linked to it in nature (e.g., in the genome of a wild-type (WT) plant) or is operably linked to it at a different site or location in nature (e.g., in the genome of a WT plant), such as a promoter.

[0093] Preferably, in the context of a nucleic acid molecule or DNA (e.g., NEENA), the term "heterologous" refers to a nucleic acid molecule that is operably linked to or manipulated to become operably linked to a second nucleic acid molecule that is not operably linked to it in nature, such as a promoter.

[0094] A heterologous expression construct comprising a nucleic acid molecule and one or more regulatory nucleic acid molecules (such as a promoter or a transcription termination signal) linked thereto is, for example, a construct derived from experimental manipulation, in which construct, a) the nucleic acid molecule or b) the regulatory nucleic acid molecule or c) both (i.e., (a) and (b)) are not in their natural (original) genetic environment or have been modified by experimental manipulation, examples of which are substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment refers to the natural chromosomal locus in the source organism or to what is present in a genomic library. In the case of a genomic library, preferably, the natural genetic environment of the sequence of the nucleic acid molecule is retained, at least in part. This environment is distributed on at least one side of the nucleic acid sequence and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1,000 bp, very particularly preferably at least 5,000 bp. A naturally occurring expression construct, for example, the natural combination of a promoter and a corresponding gene, becomes a transgenic expression construct when modified by a non-natural synthetic "artificial" method such as mutagenesis. Such methods have been described (US 5,565,350; WO 00 / 15815). For example, a nucleic acid molecule encoding a protein that is operably linked to a promoter is considered heterologous with respect to the promoter, where the promoter is not the natural promoter of the nucleic acid molecule. Preferably, the heterologous DNA is not endogenous or not naturally associated with the cell into which the heterologous DNA is introduced, but has been obtained from another cell or has been synthesized. Heterologous DNA also includes endogenous DNA sequences containing some modifications, non-naturally occurring multiple copies of endogenous DNA sequences, or such DNA sequences that are not naturally joined to another DNA sequence physically linked to the said DNA sequence. Generally, although not necessarily, the heterologous DNA encodes an RNA or a protein that is not normally encoded by the cell into which the heterologous DNA is introduced.

[0095] The term "hybridization" as defined herein is the process in which substantially complementary nucleotide sequences anneal to each other. The hybridization process can be carried out entirely in solution, i.e., both complementary nucleic acids are in solution. The hybridization process can also occur when one of the complementary nucleic acids is immobilized to a medium such as magnetic beads, agarose gel beads or any other resin. The hybridization process can also be carried out when one of the complementary nucleic acids is immobilized to a solid support such as a nitrocellulose membrane or a nylon membrane or is immobilized to a support, including but not limited to a silicate glass support (the latter being referred to as a nucleic acid array or a microarray or a nucleic acid chip), by, for example, photolithography. For hybridization to occur, the nucleic acid molecules are usually heat-denatured or chemically denatured to unwind the double-strands into two single-strands and / or to remove hairpins or other secondary structures from the single-stranded nucleic acids.

[0096] The formation or melting of the hybrid molecule depends on a variety of parameters, including but not limited to temperature. An increase in temperature favors melting, while a decrease in temperature favors hybridization. However, this process of hybrid molecule formation does not vary linearly with the applied temperature: the hybridization process is dynamic, and the nucleotide pairs that have already formed also support the pairing of adjacent nucleotides. Thus, to a good approximation, hybridization is a yes or no process, and there is a temperature that essentially defines the boundary between hybridization and non-hybridization. This temperature is the melting temperature (Tm). Tm is the temperature in degrees Celsius at which 50% of all molecules with a given nucleotide sequence hybridize into double strands and 50% exist as single strands.

[0097] The melting temperature (Tm) depends on the physical properties of the nucleic acid sequence being analyzed and can thus indicate the relationship between two different sequences. However, the melting temperature (Tm) is also affected by a variety of other parameters that are completely unrelated to the sequence, and the experimental conditions of the applied hybridization must be taken into account. For example, increasing the salt (e.g., monovalent cations) results in a higher Tm.

[0098] The Tm for a given hybridization condition can be determined by performing a physical hybridization experiment, but the Tm for a given pair of DNA sequences can also be estimated by computer simulation. In this embodiment, the equation of Meinkoth and Wahl (Anal. Biochem., 138:267–284, 1984) is used for fragments with a length of 50 bases or more: Tm = 81.5 °C + 16.6(log M) + 0.41(%GC) - 0.61(% formamide) - 500 / L.

[0099] M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine in the DNA fragment, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid molecule in base pairs. This equation is used for a salt range of 0.01 to 0.4 M and a GC% of 30% to 75%.

[0100] Although the above Tm is the temperature for a perfectly matched probe, for every 1% mismatch, the Tm decreases by approximately 1 °C (Bonner et al., J. Mol. Biol. 81:123-135, 1973): Tm = [81.5 °C + 16.6(log M) + 0.41(%GC) - 0.61(% formamide) - 500 / L] - % non-identity.

[0101] This equation can be used for probes with 35 or more nucleotides and is widely applied in scientific method literature (e.g., cited in: "Recombinant DNA Principles and Methodologies", James Greene, chapter "Biochemistry of Nucleic acids", Paul S. Miller, page 55; 1998, CRC Press), many patent applications (e.g., cited in: US 7026149) and also in data files of commercial enterprises (e.g., "Equations for Calculating Tm" from www.genomics.agilent.com).

[0102] Other formulas for calculating Tm that are less preferred in this embodiment may only be used for the indicated situations:

[0103] For DNA-RNA hybrid molecules (Casey, J. and Davidson, N. (1977) Nucleic Acids Res., 4:1539):

[0104] Tm = 79.8 °C + 18.5(log M) + 0.58(%GC) + 11.8(%GC * %GC) - 0.5(formamide %) - 820 / L.

[0105] For RNA-RNA hybrid molecules (Bodkin, D.K. and Knudson, D.L. (1985) J. Virol. Methods, 10:45):

[0106] Tm = 79.8 °C + 18.5(log M) + 0.58(%GC) + 11.8(%GC * %GC) - 0.35(formamide %) - 820 / L.

[0107] For oligonucleotide probes less than 20 bases in length (Wallace, R.B. et al. (1979) Nucleic Acid Res. 6:3535): Tm = 2 x n(A+T) + 4 x n(G+C), where n is the number of corresponding bases in the probe forming the hybrid molecule.

[0108] For oligonucleotide probes of 20 - 35 nucleotides, the modified Wallace calculation can be applied: Tm = 22 + 1.46n(A+T) + 2.92n(G+C), where n is the number of corresponding bases in the probe forming the hybrid molecule.

[0109] For other oligonucleotides, the nearest neighbor model for calculating the melting temperature should be used together with appropriate thermodynamic data:

[0110] Tm = (∑(ΔHd) + ΔHi) / (∑(ΔSd) + ΔSi + ΔSself + R × ln(cT / b)) + 16.6 log[Na+] – 273.15 (Breslauer, K. J., Frank, R., H., Marky, L. A. 1986 Predicting DNA duplex stability from the base sequence (Predicting DNA duplex stability from the base sequence). Proc. Natl. Acad. Sci. USA 83 3746–3750; Alejandro Panjkovich, Francisco Melo, 2005. Comparison of different melting temperature calculation methods for short DNA sequences (Comparison of different melting temperature calculation methods for short DNA sequences) Bioinformatics, 21(6):711-722)

[0111] Where:

[0112] Tm is the melting temperature in degrees Celsius;

[0113] ∑(ΔHd) and ∑(ΔSd) (correspondingly) are the total enthalpy sum and entropy sum calculated for all internal nearest-neighbor doublets;

[0114] ΔSself is the entropy penalty for self-complementary sequences;

[0115] ΔHi and ΔSi are the total starting enthalpy and starting entropy, respectively;

[0116] R is the gas constant (fixed at 1,987 cal / K·mol);

[0117] cT is the total strand concentration in molar units;

[0118] For non-self-complementary sequences, the constant b takes the value 4, or for duplexes of self-complementary strands or duplexes when one of the strands is in significant excess, it equals 1.

[0119] Thermodynamic calculations assume that renaturation occurs at a pH close to 7.0 in a buffer solution and a two-state transition occurs.

[0120] The thermodynamic values used for this calculation can be obtained from Table 1 of (Alejandro Panjkovich, Francisco Melo, 2005. Comparison of different melting temperature calculation methods for short DNA sequences. Bioinformatics, 21(6): 711 - 722), or from the original research papers (Breslauer, K.J., Frank, R., H., Marky, L.A. 1986 Predicting DNA duplex stability from the base sequence. Proc. Natl Acad. Sci. USA 83 3746–3750; Santa Lucia, J., Jr, Allawi, H.T., Seneviratne, P.A. 1996 Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35 3555–3562; Sugimoto, N., Nakano, S., Yoneyama, M., Honda, K. 1996 Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res. 24 4501–4505).

[0121] For computer simulation to estimate Tm according to this embodiment, first, a set of bioinformatics sequence alignment results is generated between two sequences. Such alignment results can be produced by various tools known to those skilled in the art, such as the program "Blast" (NCBI), "Water" (EMBOSS), or "Matcher" (EMBOSS) that produce local alignment results, or the program "Needle" (EMBOSS) that produces global alignment results. These tools should be applied with their default parameter sets and also with certain parameter variations. For example, the program "MATCHER" can be applied with various parameters for gap opening / gap extension (such as 14 / 4; 14 / 2; 14 / 5; 14 / 8; 14 / 10; 20 / 2; 20 / 5; 20 / 8; 20 / 10; 30 / 2; 30 / 5; 30 / 8; 30 / 10; 40 / 2; 40 / 5; 40 / 8; 40 / 10; 10 / 2; 10 / 5; 10 / 8; 10 / 10; 8 / 2; 8 / 5; 8 / 8; 8 / 10; 6 / 2; 6 / 5; 6 / 8; 6 / 10) and the program "WATER" can be applied with various parameters for gap opening / gap extension (such as 10 / 0.5; 10 / 1; 10 / 2; 10 / 3; 10 / 4; 10 / 6; 15 / 1; 15 / 2; 15 / 3; 15 / 4; 15 / 6; 20 / 1; 20 / 2; 20 / 3; 20 / 4; 20 / 6; 30 / 1; 30 / 2; 30 / 3; 30 / 4; 30 / 6; 45 / 1; 45 / 2; 45 / 3; 45 / 4; 45 / 6; 60 / 1; 60 / 2; 60 / 3; 60 / 4; 60 / 6), and these programs should also be applied by using the nucleotide sequences as given and also with one of the sequences in its reverse complementary form. For example, BlastN (NCBI) can be applied with an increased e-value cut-off (such as e+1 or even e+10) to also identify very short alignment results, particularly in small-sized databases.

[0122] It is important to consider local alignments because hybridization may not necessarily occur over the entire length of the two sequences, but may occur optimally in different regions, which then determine the actual melting temperature. Therefore, from all the generated alignment results, the alignment length, the GC content % of the alignment result (in a more precise way, the %GC content of the matching bases within the alignment result), and the alignment identity must be determined. Subsequently, the predicted melting temperature (Tm) must be calculated for each alignment result. The highest calculated Tm is used to predict the actual melting temperature.

[0123] The term "hybridize within the full sequence scope of the present invention" as defined herein means that when the sequences of the present invention are fragmented into small pieces of about 300 to 500 base lengths, for sequences with a length exceeding 300 bases, each fragment must hybridize. For example, DNA can be fragmented into small pieces by using a restriction enzyme or a combination of restriction enzymes. The bioinformatics computer simulation calculation of Tm is performed only for each fragment by the same procedure as described above. The actual hybridization of each fragment can be analyzed by standard DNA blot analysis or comparable methods known to those skilled in the art.

[0124] The term "stringency" as defined herein describes the ease by which a hybrid molecule can be formed between two nucleotide sequences. Conditions of "higher stringency" require more bases of one sequence to pair with the other sequence (the melting temperature Tm decreases under conditions of "higher stringency"), and conditions of "lower stringency" allow some more bases not to pair. Thus, the degree of relationship between two sequences can be estimated by the actual stringency conditions under which they can still form a hybrid molecule. Increasing stringency can be achieved by keeping the experimental hybridization temperature constant and decreasing the salt concentration, or by keeping the experimental hybridization temperature constant and increasing the experimental hybridization temperature or a combination of these parameters. Additionally, increasing formamide will increase stringency. Those skilled in the art are aware of additional parameters that can be varied during hybridization and the stringency conditions that are to be maintained or changed (Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and annual updates)).

[0125] Common hybridization experiments are carried out through an initial hybridization step followed by one to several washing steps. The solutions used for these steps can contain additional components such as EDTA, SDS, fragmented sperm DNA or similar reagents that prevent degradation of the analyzed sequences and / or prevent non-specific background binding of the probes, and these components are known to those skilled in the art (Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, CSH, New York or Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and annual updates)).

[0126] Common probes for hybridization experiments are generated by the random priming labeling method, which was originally developed by Feinberg and Vogelstein (Anal. Biochem., 132(1), 6 - 13(1983); Anal. Biochem., 137(1), 266 - 7(1984)) and is based on the hybridization of a mixture of all possible hexanucleotides to the DNA to be labeled. The labeled probe products will actually be a set of fragments with variable lengths, generally in the size range of 100 - 1000 nucleotides, and the highest fragment concentration is generally about 200 to 400 bp. The actual size range of the probe fragments ultimately used as probes in hybridization experiments can also be affected by: the parameters of the labeling method used, the subsequent purification of the generated probes (such as agarose gel), and the size of the template DNA used for labeling (before labeling, for example, a large template can be digested with a 4bp cutting enzyme (such as HaeIII) restrictively).

[0127] For the present invention, the sequences described herein are analyzed by hybridization experiments, in which the probes are generated from another sequence and such probes are generated by the standard random priming labeling method. For the present invention, the probes consist of a set of labeled oligonucleotides with a size of about 200 - 400 nucleotides. Hybridization between the sequences of the present invention and another sequence means that the hybridization of the probes occurs within the full sequence range of the present invention as defined above. The hybridization experiment is carried out by achieving the highest stringency according to the stringency of the final washing step. The final washing step has stringency conditions comparable to the following washing conditions: at least washing condition 1: 1.06x SSC, 0.1% SDS, 0% formamide at 50°C; in another embodiment at least washing condition 2: 1.06x SSC, 0.1% SDS, 0% formamide at 55°C; in another embodiment at least washing condition 3: 1.06x SSC, 0.1% SDS, 0% formamide at 60°C; in another embodiment at least washing condition 4: 1.06x SSC, 0.1% SDS, 0% formamide at 65°C; in another embodiment at least washing condition 5: 0.52x SSC, 0.1% SDS, 0% formamide at 65°C; in another embodiment at least washing condition 6: 0.25x SSC, 0.1% SDS, 0% formamide at 65°C; in another embodiment at least washing condition 7: 0.12x SSC, 0.1% SDS, 0% formamide at 65°C; in another embodiment at least washing condition 8: 0.07x SSC, 0.1% SDS, 0% formamide at 65°C.

[0128] "Low stringency washing" has a stringency condition that is comparable to the stringency condition of at least washing condition 1, but is not more stringent than washing condition 3, where the washing conditions are as described above.

[0129] "High stringency washing" has a stringency condition that is comparable to at least washing condition 4, in another embodiment at least washing condition 5, in another embodiment at least washing condition 6, in another embodiment at least washing condition 7, in another embodiment at least washing condition 8, where the washing conditions are as described above.

[0130] "Identity": When comparing two or more nucleic acid or amino acid molecules, "identity" means that the sequences of the molecules have a certain degree of sequence similarity, i.e., the sequences are partially identical.

[0131] Enzyme variants can be defined by their sequence identity compared to the parental enzyme. Sequence identity is usually provided as "sequence identity %" or "identity %". To determine the percentage of identity between two amino acid sequences, in the first step, a pairwise sequence alignment result is generated between the two sequences, where the two sequences are aligned over their entire length (i.e., a pairwise global alignment). Using a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443 - 453), preferably by using the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)), with the program default parameters (gap opening = 10.0, gap extension = 0.5, and matrix = EDNAFULL), the alignment result is generated.

[0132] The following example is intended to illustrate two nucleotide sequences, but the same calculation applies to protein sequences:

[0133] Seq A: AAGATACTG Length: 9 bases

[0134] Seq B: GATCTGA Length: 7 bases

[0135] Therefore, the shorter sequence is sequence B.

[0136] Generating a pairwise global alignment showing the two sequences over their entire length results in

[0137]

[0138] The "I" symbol in the alignment result indicates identical residues (which means the bases of DNA or the amino acids of a protein). The number of identical residues is 6.

[0139] The "-" symbol in the alignment result indicates a gap. The number of gaps introduced by alignment within Seq B is 1. The numbering of the gaps introduced by alignment is 2 at the boundary of Seq B and 1 at the boundary of Seq A.

[0140] The alignment length showing the aligned sequences over their entire length is 10.

[0141] Pairwise alignments generated according to the present invention that show the shorter sequence over its entire length thus result in:

[0142]

[0143] Pairwise alignments generated according to the present invention that show Sequence A over its entire length thus result in:

[0144]

[0145] Pairwise alignments generated according to the present invention that show Sequence B over its entire length thus result in:

[0146]

[0147] The alignment length showing the shorter sequence over its entire length is 8 (there is a gap considered in the alignment length of the shorter sequence).

[0148] Therefore, the alignment length showing Seq A over its entire length will be 9 (meaning Seq A is the sequence of the present invention).

[0149] Therefore, the alignment length showing Seq B over its entire length will be 8 (meaning Seq B is the sequence of the present invention).

[0150] After aligning the two sequences, in the second step, the identity value is determined from the resulting alignment. For the purposes of this specification, the percentage identity is calculated as follows: Identity % = (number of identical residues / length of the alignment region showing the corresponding sequence of the present invention over its entire length) * 100. Thus, according to this embodiment, the sequence identity related to comparing two amino acid sequences is calculated by dividing the number of identical residues by the length of the alignment region showing the corresponding sequence of the present invention over its entire length. This value is multiplied by 100 to obtain the "Identity %". According to the example provided above, Identity %: for Seq A as the sequence of the present invention, (6 / 9) * 100 = 66.7%; for Seq B as the sequence of the present invention, (6 / 8) * 100 = 75%.

[0151] Indel (insertion / deletion) is a term for the random insertion or deletion of bases related to NHEJ repair of DSBs in the genome of an organism. It is classified among small genetic variations and measures from 1 to 10,000 base pairs in length. As used herein, it refers to the random insertion or deletion of bases in or immediately adjacent to the target site (e.g., less than 1000bp, 900bp, 800bp, 700bp, 600bp, 500bp, 400bp, 300bp, 250bp, 200bp, 150bp, 100bp, 50bp, 40bp, 30bp, 25bp, 20bp, 15bp, 10bp, or 5bp upstream and / or downstream thereof).

[0152] For the introduction of a donor DNA molecule into a target site of a target DNA, terms such as "introduce", "import", etc. mean the introduction of the sequence of the donor DNA molecule into any of the target regions, for example, by physically integrating the donor DNA molecule or a portion thereof into the target region, or the introduction of the sequence of the donor DNA molecule or a portion thereof into the target region where the donor DNA is used as a polymerase template.

[0153] Isogenic: Organisms (e.g., plants) that are genetically identical except for potentially differing by the presence or absence of heterologous DNA sequences.

[0154] Isolated: As used herein, the term "isolated" means that a material has been removed by human intervention and exists outside of its original natural environment and is thus not a product of nature. An isolated material or molecule (such as a DNA molecule or an enzyme) can exist in a purified form or can exist in a non-natural environment such as, for example, in a transgenic host cell. For example, a naturally occurring polynucleotide or polypeptide present in a living plant is not isolated, whereas the same polynucleotide or polypeptide separated from some or all of the coexisting materials in that natural system is isolated. Such polynucleotides can be part of a vector and / or such polynucleotides or polypeptides can be part of a composition, and are isolated because the vector or composition is not part of its original environment. Preferably, the term "isolated", when used in connection with a nucleic acid molecule, as in "isolated nucleic acid sequence", refers to a nucleic acid sequence that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in its natural source. An isolated nucleic acid molecule is a nucleic acid molecule that exists in a form or environment different from that in which it is found in nature. In contrast, an unisolated nucleic acid molecule is a nucleic acid molecule found in the state in which it exists in nature, such as DNA and RNA. For example, a given DNA sequence (e.g., a gene) is found adjacent to neighboring genes on a host cell chromosome; an RNA sequence, such as a particular mRNA sequence encoding a specific protein, is found in a cell as a mixture with numerous other mRNAs encoding a variety of proteins. However, an isolated nucleic acid sequence comprising, for example, SEQ ID NO:12 includes, by way of example, such nucleic acid sequences that are ordinarily found in a cell containing SEQ ID NO:12, where the nucleic acid sequence is in a chromosomal location different from that of the natural cell or an extrachromosomal location or is otherwise flanked by nucleic acid sequences different from those found in nature. An isolated nucleic acid sequence can exist in single-stranded or double-stranded form. When an isolated nucleic acid sequence is used to express a protein, the nucleic acid sequence will contain at least a portion of the sense strand or coding strand (i.e., the nucleic acid sequence can be single-stranded). Alternatively, it can contain the sense strand and the antisense strand (i.e., the nucleic acid sequence can be double-stranded).

[0155] Non-coding: The term "non-coding" refers to a sequence in a nucleic acid molecule that does not encode some or all of the expressed protein. Non-coding sequences include, but are not limited to, introns, enhancers, promoter regions, 3' untranslated regions, and 5' untranslated regions.

[0156] Nucleic Acids and Nucleotides: The terms "nucleic acid" and "nucleotide" refer to naturally occurring or synthetic or artificial nucleic acids or nucleotides. The terms "nucleic acid" and "nucleotide" include deoxyribonucleotides or ribonucleotides or any nucleotide analogs and polymers or hybrids thereof in single-stranded or double-stranded, sense or antisense forms. Unless otherwise specified, a particular nucleic acid sequence also inherently includes variants (e.g., degenerate codon substitutions) and complementary sequences modified in a conservative manner, as well as the explicitly recited sequences. The term "nucleic acid" is used interchangeably herein with "gene", "cDNA", "mRNA", "oligonucleotide", and "polynucleotide". Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar, and / or phosphate ester, including but not limited to 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines of cytosine, 5-bromo-uracil substitution, etc.; and 2'-position sugar modifications, including but not limited to ribonucleotides with sugar modifications where the 2'-OH is replaced by a group selected from H, OR, halogen, SH, SR, NH2, NHR, NR2, or CN. Short hairpin RNAs (shRNAs) may also contain non-natural elements such as non-natural bases, e.g., inosine and xanthine, non-natural sugars, e.g., 2'-methoxyribose, or non-natural phosphodiester bonds, e.g., methylphosphonates, phosphorothioates, and peptides.

[0157] Nucleic Acid Sequence: The phrase "nucleic acid sequence" refers to a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotides read from the 5'-end to the 3'-end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA, and DNA or RNA that mainly plays a structural role. "Nucleic acid sequence" also refers to a continuous string of abbreviations, letters, characters, or words representing nucleotides. In one embodiment, the nucleic acid can be a "probe", which is a relatively short nucleic acid, usually less than 100 nucleotides in length. Frequently, nucleic acid probes have a length of about 50 nucleotides to about 10 nucleotides. The "target region" of a nucleic acid is the portion of the nucleic acid identified as the target. The "coding region" of a nucleic acid is the portion of the nucleic acid that, when placed under the control of appropriate regulatory sequences, is transcribed and translated in a sequence-specific manner to produce a specific polypeptide or protein. This coding region is said to encode such a polypeptide or protein.

[0158] Oligonucleotide: The term "oligonucleotide" refers to oligomers or polymers of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, as well as oligonucleotides having non-naturally occurring moieties that function similarly. Such modified or substituted oligonucleotides are often preferred over their native forms due to desirable properties such as enhanced cellular uptake, enhanced nucleic acid target affinity, and increased stability in the presence of nucleases. Oligonucleotides preferably comprise two or more nucleomonomers covalently coupled to each other by a bond (e.g., a phosphodiester bond) or a substitute linkage.

[0159] Overhang: An "overhang" is a relatively short single-stranded nucleotide sequence (also referred to as an "extension", "protrusion", or "sticky end") at the 5'- or 3'-hydroxyl end of a double-stranded oligonucleotide molecule.

[0160] Polypeptide: The terms "polypeptide", "peptide", "oligopeptide", "polypeptide", "gene product", "expression product", and "protein" are used interchangeably herein to refer to polymers or oligomers of contiguous amino acid residues.

[0161] Preprotein: A protein that is normally targeted to an organelle such as a chloroplast and still contains its transit peptide.

[0162] With respect to introducing a donor DNA molecule into a target region, "precise" means introducing the sequence of the donor DNA molecule into the target region without any insertions / deletions, duplications, or other mutations as compared to the unaltered DNA sequence of the target region that is not included in the sequence of the donor DNA molecule.

[0163] Primary transcript: The term "primary transcript" as used herein refers to an immature RNA transcript of a gene. A "primary transcript" still contains introns and / or still does not contain a polyadenylate tail or a cap structure and / or is lacking other modifications necessary for its normal functioning as a transcript, such as trimming or editing.

[0164] A "promoter" or "promoter sequence" or "regulatory nucleic acid" is a nucleotide sequence located upstream of a gene on the same strand as the gene that enables transcription of the gene. The promoter is followed by the transcription start site of the gene. The promoter is recognized by RNA polymerase (along with any required transcription factors), which initiates transcription. A functional fragment or functional variant of a promoter is a nucleotide sequence that can be recognized by RNA polymerase and is capable of initiating transcription.

[0165] Purified: As used herein, the term "purified" refers to a molecule, i.e., a nucleic acid sequence or an amino acid sequence, that has been removed, separated or isolated from its natural environment. A "substantially purified" molecule is at least 60% free, preferably at least 75% free and more preferably at least 90% free of other components with which it is naturally associated. A purified nucleic acid sequence can be an isolated nucleic acid sequence.

[0166] Recombinant: In the context of nucleic acid molecules, the term "recombinant" refers to nucleic acid molecules produced by recombinant DNA techniques. Recombinant nucleic acid molecules can also include molecules that do not exist in nature but have been modified, altered, mutated or engineered by man. Preferably, a "recombinant nucleic acid molecule" is a non-naturally occurring nucleic acid molecule that differs from a naturally occurring nucleic acid molecule by at least one nucleic acid in sequence. A "recombinant nucleic acid molecule" can also include a "recombinant construct" that contains a series of nucleic acid molecules that do not naturally occur in this order and are preferably operably linked. Preferred methods for producing the recombinant nucleic acid molecules can include cloning techniques, directed or non-directed mutagenesis, synthesis or recombination techniques.

[0167] Reduced expression: As used interchangeably herein, "reduce" or "decrease" the expression of a nucleic acid molecule in a cell, and this means that the expression level of the nucleic acid molecule in the cell after application of the method of the present invention is lower than its expression in the cell before the method, or lower than that in a reference cell lacking the recombinant nucleic acid molecule of the present invention. For example, the reference cell contains the same construct, which contains the starting regulatory nucleic acid molecule of the present invention and does not contain the synthetic regulatory nucleic acid molecule of the present invention. As used herein, the terms "reduce" or "decrease" are synonymous and mean herein that the expression of the nucleic acid molecule to be expressed is reduced, preferably significantly reduced. As used herein, "reducing" the level of a substance (such as a protein, mRNA or RNA) means that the level is decreased relative to substantially the same cells cultured under substantially the same conditions and lacking the recombinant nucleic acid molecule of the present invention (e.g., containing the starting regulatory nucleic acid molecule of the present invention and not containing the synthetic regulatory nucleic acid molecule of the present invention). As used herein, a decrease in the level of a substance (such as preRNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by a target gene) and / or the level of a protein product encoded by a target gene means that the level is decreased by 10% or more, such as 20% or more, 30% or more, 40% or more, preferably 50% or more, such as 60% or more, 70% or more, 80% or more, 90% or more, relative to a cell lacking the recombinant nucleic acid molecule of the present invention (e.g., containing the starting regulatory nucleic acid molecule of the present invention and not containing the synthetic regulatory nucleic acid molecule of the present invention). The reduction can be determined by methods familiar to those skilled in the art. Thus, for example, the reduction in the amount of nucleic acid or protein can be determined by immunological detection methods of proteins. Additionally, techniques such as protein assays, fluorescence assays, RNA hybridization assays, nuclease protection assays, reverse transcription (quantitative RT-PCR), ELISA (enzyme-linked immunosorbent assay), Western blotting, radioimmunoassay (RIA) or other immunoassays and fluorescence-activated cell analysis (FACS) can be used to measure a specific protein or RNA in a cell. Depending on the type of protein product that has been reduced, its activity or the effect on a biological or cellular phenotype can also be determined. Methods for determining the amount of protein are known to those skilled in the art. Examples that can be mentioned are: the micro Biuret method (Goa J (1953) Scand J Clin Lab Invest 5:218-222), the Folin-Ciocalteau method (Lowry OH et al. (1951) J Biol Chem 193:265-275) or measuring the absorbance of CBB G-250 (Bradford MM (1976) Analyt Biochem 72:248-254).

[0168] Sense: The term "sense" is understood to mean a nucleic acid molecule that has a sequence complementary or identical to a target sequence, for example a sequence that binds to a protein transcription factor and is involved in the expression of a given gene. According to a preferred embodiment, the nucleic acid molecule comprises a gene of interest and elements that permit the expression of the gene of interest.

[0169] Significant increase or decrease: An increase or decrease greater than the margin of error inherent in the measurement technique, for example in enzyme activity or in gene expression, preferably an increase or decrease in the activity of a control enzyme or the expression in a control cell of about 2-fold or more, more preferably an increase or decrease of about 5-fold or more, and most preferably an increase or decrease of about 10-fold or more.

[0170] Small nucleic acid molecule: "Small nucleic acid molecule" is understood to mean a molecule composed of nucleic acid or its derivatives such as RNA or DNA. They can be double-stranded or single-stranded, and their length is between about 15 and about 30 bp, for example between 15 and 30 bp, more preferably between about 19 and about 26 bp, for example between 19 and 26 bp, even more preferably between about 20 and about 25 bp, for example between 20 and 25 bp. In a particularly preferred embodiment, the length of the oligonucleotide is between about 21 and about 24 bp, for example between 21 and 24 bp. In the most preferred embodiment, the length of the small nucleic acid molecule is about 21 bp and about 24 bp, for example 21 bp and 24 bp.

[0171] Substantially complementary: In the broadest sense, as used herein with respect to a nucleotide sequence relative to a reference or target nucleotide sequence, the term "substantially complementary" means a nucleotide sequence having at least 60%, more desirably at least 70%, more desirably at least 80% or 85%, preferably at least 90%, more preferably at least 93%, still more preferably at least 95% or 96%, yet still more preferably at least 97% or 98%, still more preferably at least 99% or most preferably 100% identity percentage (the latter being equivalent to the term "identical" in this context) between the substantially complementary nucleotide sequence and the complete complementary sequence of the reference or target nucleotide sequence. Preferably, identity is evaluated against the reference nucleotide sequence over a length of at least 19 nucleotides, preferably at least 50 nucleotides, more preferably over the entire length of the nucleic acid sequence (if not, it is otherwise stated below). Using the default GAP analysis of GCG from the University of Wisconsin, the SEQWEB application of GAP, sequence comparison is performed based on the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453; as defined above). A nucleotide sequence "substantially complementary" to a reference nucleotide sequence hybridizes to the reference nucleotide sequence under low stringency conditions, preferably medium stringency conditions, most preferably high stringency conditions (as defined above).

[0172] As used herein, "target region" means a region that is located at a distance, for example, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 60 bases, 70 bases, 80 bases, 90 bases, 100 bases, 125 bases, 150 bases, 200 bases, or 500 bases or more bases from a target site or that contains a target site into which the sequence of a donor DNA molecule is to be introduced into the cell genome.

[0173] As used herein, "target site" means a position in the genome where double-strand breaks or one or a pair of single-strand breaks (nicks) are induced using recombinant techniques such as Zn fingers, TALENs, restriction enzymes, homing endonucleases, RNA-guided nucleases, RNA-guided nickases such as CRISPR / Cas nucleases or nickases, etc.

[0174] Transgene: As used herein, the term "transgene" refers to any nucleic acid sequence that is introduced into the genome of a cell by experimental manipulation. A transgene can be an "endogenous DNA sequence" or a "heterologous DNA sequence" (i.e., "foreign DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence that naturally occurs in the cell into which the nucleotide sequence is introduced, provided that it does not contain some modifications relative to the naturally occurring sequence (e.g., for example, point mutations, the presence of selectable marker genes, etc.).

[0175] Transgenic: When referring to an organism, the term "transgenic" means that the organism is transformed with a recombinant DNA molecule, preferably stably transformed, wherein the recombinant DNA molecule preferably contains a suitable promoter operably linked to a DNA sequence of interest.

[0176] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a genomic integration vector, or "integration vector", which can integrate into the chromosomal DNA of a host cell. Another type of vector is an episomal vector, i.e., a nucleic acid molecule capable of extrachromosomal replication. A vector capable of directing the expression of a gene operably linked to it is referred to herein as an "expression vector". In this specification, "plasmid" and "vector" are used interchangeably unless the context dictates otherwise. Expression vectors designed to produce the RNAs described herein in vitro or in vivo can contain sequences recognized by any RNA polymerase, including mitochondrial RNA polymerase, RNA pol I, RNA pol II, and RNA pol III. These vectors can be used to transcribe the desired RNA molecules in the cells according to the present invention. Brief Description of the Drawings

[0178] Figure 1

[0179] The plasmid map of the single CRISPR / Cas9 plasmid pCC009 is described. Plasmid pCC009 is a derivative of plasmid pJOE8999.1, which carries the spacer of the Bacillus licheniformis amyB gene and the DNA donor sequences HomA and HomB at the 5' and 3' of the amyB gene, respectively. PmanP: promoter of the Bacillus subtilis manP gene; pUC ORI: high-copy replication origin of Escherichia coli; kanamycin resistance gene functional in both Bacillus and Escherichia coli; rep pE194: fragment of plasmid pE194 that confers temperature-sensitive plasmid replication in Bacillus; PvanP: promoter driving the expression of spacer-sgRNA (crRNA repeat + 'gRNA); T0 terminator from λ; t1t2 terminator from the Escherichia coli rrnB gene; HomA and HomB: 5' and 3' sequences of the amyB gene fused together for gene deletion; Cas9: Cas9 endonuclease from Streptococcus pyogenes.

[0180] Figure 2 :

[0181] Sequence alignment showing selected regions of the mutated promoter sequences – reference is made to nt 15 to nt 128 for the promoter sequences PV4 (SEQ ID 028) and PV8 (SEQ ID 029). Inside the reference promoter sequences of the PV4 (SEQ ID 028) and PV8 (SEQ ID 029) promoters, the -35 region, the -10 region, the transcription start site (TSS) and the Shine Dalgarno sequence (SD) are described in italic letters and shaded in grey. Nucleotide deletions, insertions and mutations are described in bold.

[0182] Figure 3

[0183] Analysis of the Bacillus licheniformis amyB gene deletion in single colonies by colony PCR with oligonucleotides SEQ ID 009 and SEQ ID 010 located outside the homology regions for gene deletion. The gene deletion efficiency of the Bacillus licheniformis amylase amyB gene (as the percentage of clones with an inactivated amylase gene relative to a total of 20 clones analyzed for each gene deletion construct) was plotted for each gene deletion construct as shown. A. Describes the relative deletion efficiency of the deletion plasmids derived from the PV4 promoter variant. B. Describes the relative deletion efficiency of the deletion plasmids derived from the PV8 promoter variant.

[0184] Figure 4

[0185] A. The gene deletion efficiency of the Bacillus licheniformis hag gene (as the percentage of clones with an inactivated hag gene relative to a total of 20 analyzed clones) was plotted for two deletion constructs and promoter variants as shown respectively. The mean of three independent experiments together with the standard deviation is shown. Analysis of the hag gene deletion by colony PCR with oligonucleotides SEQ ID 087 and SEQ ID 088 located outside the homology regions for gene deletion. B. Describes the relative mutation efficiency of two deletion constructs and promoter variants for the introduction of point mutations inside the Bacillus licheniformis degU gene, as the percentage of clones with a mutated degU gene relative to a total of 20 analyzed clones. The mean of three independent experiments together with the standard deviation is shown. Analysis of the degU gene mutation by colony PCR with oligonucleotides SEQ ID 089 and SEQ ID 090 located outside the homology regions used for introducing the gene mutation, followed by PstI restriction digestion of the PCR fragments to distinguish between the native and mutant degU gene loci.

[0186] Figure 5

[0187] A. The gene deletion efficiency of the Bacillus subtilis amylase amyE gene (as the percentage of clones with an inactivated amyE gene relative to a total of 20 analyzed clones) was plotted against two deletion constructs and promoter variants as shown. The mean of three independent experiments along with the standard deviation is shown. The gene deletion of the amyE gene was analyzed by colony PCR using oligonucleotides SEQ ID 091 and SEQ ID 092 located outside the homology region for gene deletion. B. The relative deletion efficiency of two deletion constructs and promoter variants for deleting the subtilisin aprE gene of Bacillus subtilis, respectively, is described as the percentage of clones with an inactivated aprE gene relative to a total of 20 analyzed clones. The mean of three independent experiments along with the standard deviation is shown. The gene deletion of the aprE gene was analyzed by colony PCR using oligonucleotides SEQ ID 093 and SEQ ID 094 located outside the homology region for gene deletion.

[0188] Figure 6

[0189] A. The gene deletion efficiency of the Bacillus licheniformis vpr gene (as the percentage of clones with an inactivated vpr gene relative to a total of 20 analyzed clones) was plotted against three deletion constructs and spacer variants as shown. The gene deletion of the vpr gene was analyzed by colony PCR using oligonucleotides SEQ ID 095 and SEQ ID 096 located outside the homology region for gene deletion. B. The relative deletion efficiency of three deletion constructs and spacer variants for deleting the epr gene of Bacillus licheniformis, respectively, is described as the percentage of clones with an inactivated epr gene relative to a total of 20 analyzed clones. The gene deletion of the epr gene was analyzed by colony PCR using oligonucleotides SEQ ID 097 and SEQ ID 098 located outside the homology region for gene deletion.

[0190] Figure 7

[0191] The gene integration efficiency of replacing the amyB gene of Bacillus licheniformis with the PaprE-GFPmut2 expression cassette (as the percentage of clones with an integrated PaprE-GFPmut2 expression cassette relative to a total of 20 analyzed clones) was plotted against two different Bacillus licheniformis strains Bli#005 and P308 as shown. The mean of two independent experiments along with the standard deviation is shown. The integration was analyzed by colony PCR using oligonucleotides SEQ ID 009 and SEQ ID 010 located outside the homology region for gene integration.

[0192] Figure 8

[0193] The gene deletion efficiency of the sporulation genes sigE, sigF, and spoIIE of Bacillus pumilus (as the percentage of clones with inactivated sporulation genes relative to a total of 20 clones for each analyzed sporulation gene) was plotted as shown. Gene deletion of the sigE, sigF, and spoIIE genes was analyzed by colony PCR using the oligonucleotides SEQ ID099 and SEQ ID 100, SEQ ID 101 and SEQ ID 102, and SEQ ID 103 and SEQ ID 104, respectively, which are located outside the homology regions for gene deletion. Example

[0194] Materials and methods

[0195] The following examples merely serve to illustrate the invention. Numerous possible variations that are obvious to those skilled in the art also fall within the scope of the invention.

[0196] Unless otherwise stated, the following experiments have been carried out by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering and in the fermentative production of chemical compounds by culturing 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, NY. 2001) and Chmiel et al. (Bioprocesstechnik 1. Einführung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991).

[0197] Electrocompetent Bacillus licheniformis cells and electroporation

[0198] DNA was transformed into Bacillus licheniformis strains DSM641 and ATCC53926 by electroporation. Preparation of electrocompetent Bacillus licheniformis cells and DNA transformation were carried out essentially as described by Brigidi et al. (Brigidi, P., Mateuzzi, D. (1991). Biotechnol. Techniques 5, 5), with the following modifications: once the transforming DNA was added, the cells were resuspended in 1 ml of LBSPG buffer and incubated at 37 °C for 60 minutes 1989, FEMS Microbiol. Lett., 61:165 - 170), and then plated on selective LB agar plates.

[0199] To overcome the restriction modification system specific to Bacillus licheniformis strains DSM641 and ATCC53926, plasmid DNA was isolated from Ec#098 cells as described below. For transfer into a Bacillus licheniformis restriction enzyme knockout strain, plasmid DNA was isolated from Escherichia coli INV110 cells (Life technologies).

[0200] Electrocompetent Bacillus pumilus cells and electroporation

[0201] DNA was transformed into Bacillus pumilus DSM14395 by electroporation. Preparation of electrocompetent Bacillus pumilus DSM14395 cells and DNA transformation were carried out as described for Bacillus licheniformis cells.

[0202] To overcome the restriction modification system specific to Bacillus pumilus, plasmid DNA was isolated from Escherichia coli DH10B cells and the plasmid DNA was methylated in vitro with a complete cell extract from Bacillus pumilus DSM14395 according to the method described for Bacillus licheniformis in patent DE4005025.

[0203] Electrocompetent Bacillus subtilis cells and electroporation

[0204] DNA was transformed into Bacillus subtilis ATCC6051a by electroporation as described for Bacillus licheniformis and Bacillus pumilus, respectively. Plasmid DNA isolated from Escherichia coli DH10B cells can be readily used for transfer into Bacillus subtilis.

[0205] Plasmid isolation

[0206] Plasmid DNA was isolated from Bacillus cells and Escherichia coli cells by standard molecular biology methods described in (Sambrook, J. and Russell, D. W. "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001) or the alkaline lysis method (Birnboim, H. C., Doly, J. (1979). Nucleic Acids Res 7(6):1513 - 1523). Compared with Escherichia coli, Bacillus cells were treated with 10 mg / ml lysozyme at 37 °C for 30 minutes and then the cells were lysed.

[0207] Oligonucleotides were renatured to form oligonucleotide - duplexes.

[0208] Adjust the oligonucleotides to a concentration of 100 μM in water. Add 5 μl of the forward oligonucleotide and 5 μl of the corresponding reverse oligonucleotide to 90 μl of 30 mM Hepes-buffer (pH 7.8). Heat the reaction mixture to 95 °C for 5 minutes and then perform renaturation by temperature cycling from 95 °C to 4 °C at a rate of 0.1 °C / second (Cobb, R.E., Wang, Y. and Zhao, H. (2015). High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR / Cas System. ACS Synthetic Biology, 4(6), 723–728).

[0209] Methods and Techniques in Molecular Biology

[0210] Standard methods in molecular biology, not limited to culturing Bacillus and Escherichia coli microorganisms, DNA electroporation, isolation of genomic and plasmid DNA, PCR reactions, and cloning techniques, are generally carried out as described by Sambrook and Rusell. (Sambrook, J. and Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001.)

[0211] Strain

[0212] Escherichia coli strain Ec#098

[0213] Escherichia coli strain Ec#098 is an Escherichia coli INV110 strain (Life technologies) carrying the expression plasmid pMDS003 encoding a DNA methyltransferase (WO2019016051).

[0214] Generate a Bacillus licheniformis gene k.o strain

[0215] For gene deletion in Bacillus licheniformis strains DSM641 and ATCC53926 (US5352604) and their derivatives, the deletion plasmid was transformed into Escherichia coli strain Ec#098 made competent according to the Chung method (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), and subsequently selected 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 transformation into Bacillus licheniformis strains. The isolated plasmid DNAs carried the DNA methylation patterns of Bacillus licheniformis strains DSM641 and ATCC53926 respectively and were protected from degradation when transferred into Bacillus licheniformis.

[0216] Bacillus licheniformis P304: Deletion of restriction endonucleases

[0217] Electrocompetent Bacillus licheniformis DSM641 cells (US5352604) were prepared as described above and transformed with 1 μg of the pDel006 restriction enzyme gene deletion plasmid isolated from Escherichia coli Ec#098, and subsequently plated on LB agar plates containing 5 μg / ml erythromycin at 30°C.

[0218] The gene deletion procedure was carried out as described below:

[0219] The Bacillus licheniformis cells carrying the plasmid were cultivated on an LB agar plate containing 5 μg / ml erythromycin at 45 °C, so as to drive the integration of the deletion plasmid into the chromosome by the Campbell recombination process. Meanwhile, one of the homologous regions of pDel006 was homologous to the sequence at the 5' or 3' of the aprE gene. The clones were picked out and cultivated in LB medium at 45 °C for 6 hours without selection pressure, and then plated on an LB agar plate containing 5 μg / ml erythromycin at 30 °C. The independent clones were picked out and screened for successful genomic deletion of the restriction enzyme gene by colony PCR using oligonucleotides SEQ ID 014 and SEQ ID 015. The presumptive deletion-positive independent clones were picked out and subjected to two consecutive overnight incubations in antibiotic-free LB medium at 45 °C to eliminate the plasmid, and then plated on an LB agar plate at 37 °C and incubated overnight. The successful genomic deletion of the restriction enzyme gene was analyzed by colony PCR for a single clone. The erythromycin-sensitive single clone with correct deletion of the restriction enzyme gene was isolated and named Bacillus licheniformis P304.

[0220] Bacillus licheniformis P308: Deletion of the poly-γ-glutamic acid synthesis gene

[0221] The electrocompetent Bacillus licheniformis P304 cells were prepared as described above and transformed with 1 μg of the pDel007 pga gene deletion plasmid isolated from Escherichia coli INV110 cells (Life technologies), and then plated on an LB agar plate containing 5 μg / ml erythromycin at 30 °C.

[0222] The gene deletion process was carried out as described for the deletion of the restriction enzyme gene.

[0223] The deletion of the pga gene was analyzed by PCR using oligonucleotides SEQ ID 017 and SEQ ID 018. The resulting Bacillus licheniformis strain with the pga synthesis gene deleted was named Bacillus licheniformis P308.

[0224] Bacillus licheniformis Bli#002: Deletion of the aprE gene

[0225] The electrocompetent Bacillus licheniformis ATCC53926 cells were prepared as described above and transformed with 1 μg of the pDel003 aprE gene deletion plasmid isolated from Escherichia coli Ec#098, and then plated on an LB agar plate containing 5 μg / ml erythromycin at 30 °C.

[0226] The gene deletion process was carried out as described for the deletion of the restriction enzyme gene. The deletion of the aprE gene was analyzed by PCR using oligonucleotides SEQ ID 020 and SEQ ID 021. The resulting Bacillus licheniformis strain with the aprE gene deleted was named Bli#002.

[0227] Bacillus licheniformis Bli#005: Deletion of the poly-γ-glutamic acid synthesis gene

[0228] In Bacillus licheniformis Bli#002, the poly-γ-glutamic acid synthesis gene was deleted as described for the deletion of the pga gene in Bacillus licheniformis P304, except that the pDel007 plasmid was isolated from Escherichia coli Ec#098 cells. The resulting strain was named Bli#005.

[0229] Plasmid

[0230] pEC194RS - Bacillus temperature-sensitive deletion plasmid.

[0231] The plasmid pE194 was PCR-amplified with oligonucleotides SEQ ID 001 and SEQ ID 002 flanked by PvuII sites, digested with the restriction endonuclease PvuII and ligated into the vector pCE1 digested with the restriction enzyme SmaI. pCE1 is a pUC18 derivative in which the BsaI site within the ampicillin resistance gene has been removed by silent mutation. The ligation mixture was transformed into Escherichia coli DH10B cells (Life technologies). The transformants were spread and incubated overnight at 37 °C on an LB agar plate containing 100 μg / ml ampicillin. Plasmid DNA was isolated from independent clones and the correctness was analyzed by restriction digestion. The resulting plasmid was named pEC194S.

[0232] The type II assembled mRFP cassette containing the additional nucleotide restriction site BamHI was 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) using oligonucleotides SEQ ID 003 and SEQ ID 004. The PCR fragment and pEC194S were restricted with the restriction enzyme BamHI, then ligated and transformed into Escherichia coli DH10B cells (Life technologies). The transformants were spread and incubated overnight at 37 °C on LB agar plates containing 100 μg / ml ampicillin. Plasmid DNA was isolated from independent clones and the correctness was analyzed by restriction digestion. The resulting plasmid pEC194RS carried the mRFP cassette with the open reading frame present head-to-head with the open reading frame of the erythromycin resistance gene.

[0233] pDel003 – aprE gene deletion plasmid

[0234] The gene deletion plasmid for the aprE gene of Bacillus licheniformis was constructed using plasmid pEC194RS and the gene synthesis construct SEQ ID 019 of the genomic regions containing the 5' and 3' of the aprE gene, with BsaI sites compatible with pEC194RS flanking the genomic regions. Type II assembly using the restriction endonuclease BsaI was carried out as described (Radeck, J., Meyer, D., Lautenschlager, N. and Mascher, T. 2017. Bacillus SEVA siblings: A GoldenGate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7:14134) and the reaction mixture was subsequently transformed into Escherichia coli DH10B cells (Life technologies). The transformants were spread and incubated overnight at 37 °C on LB agar plates containing 100 μg / ml ampicillin. Plasmid DNA was isolated from independent clones and the accuracy was analyzed by restriction digestion. The resulting aprE deletion plasmid was named pDel003.

[0235] pDel006 – Restriction enzyme gene deletion plasmid

[0236] The gene deletion plasmid for the restriction enzyme gene (SEQ ID 012) in the Bacillus licheniformis DSM641 restriction modification system (SEQ ID 011) was constructed using plasmid pEC194RS and the gene synthesis construct SEQ ID 013 of the genomic regions containing the 5' and 3' of the restriction enzyme gene, with BsaI sites compatible with pEC194RS flanking the genomic regions. Type II assembly using the restriction endonuclease BsaI was carried out as described above and the reaction mixture was subsequently transformed into Escherichia coli DH10B cells (Life technologies). The transformants were spread and incubated overnight at 37 °C on LB agar plates containing 100 μg / ml ampicillin. Plasmid DNA was isolated from independent clones and the accuracy was analyzed by restriction digestion. The resulting restriction enzyme deletion plasmid was named pDel006.

[0237] pDel007 – Poly-γ-glutamic acid synthesis gene deletion plasmid

[0238] A deletion plasmid for generating genes involved in the deletion of Bacillus licheniformis poly-γ-glutamate (pga) production, namely ywsC (pgsB), ywtA (pgsC), ywtB (pgsA), and ywtC (pgsE), as described in pDel006, was constructed using a gene synthesis construct SEQ ID 016, which contains genomic regions flanking the 5' and 3' sides of the ywsC, ywtA (pgsC), ywtB (pgsA), and ywtC (pgsE) genes. The genomic regions are flanked by BsaI sites compatible with pEC194RS. The resulting pga deletion plasmid was named pDel007.

[0239] Plasmid p689-T2A-lac

[0240] Plasmid p689-T2A-lac contains the lacZ-α gene flanked by BpiI restriction sites, which are further flanked by the T1 terminator of the Escherichia coli rrnB gene on the 5' side and the T0λ terminator on the 3' side, and was ordered as a gene synthesis construct (SEQ ID 073).

[0241] Plasmid p890 PaprE-GFPmut2

[0242] The promoter of the Bacillus licheniformis aprE gene (US5352604) of plasmid pCB56C was PCR amplified using oligonucleotides SEQ ID074 and SEQ ID 075. A GFPmut2 gene variant (accession number AF302837) (SEQ ID 076) with flanking BpiI restriction sites was ordered as a gene synthesis fragment (Geneart Regensburg). The gene expression construct containing the Bacillus licheniformis-derived PaprE promoter fused to the GFPmut2 variant was cloned into plasmid p689-T2A-lac by type II assembly using the 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 was subsequently transformed into electrocompetent Escherichia coli DH10B cells. The transformants were spread and incubated overnight at 37 °C on LB agar plates containing 100 μg / ml ampicillin. Plasmid DNA was isolated from independent clones and the correctness was analyzed by restriction digestion and sequencing. The resulting plasmid was named p890 PaprE-GFPmut2.

[0243] Plasmid pJOE8999.1:

[0244] Altenbuchner J. 2016. Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system. Appl Environ Microbiol 82:5421–5.

[0245] Plasmid pJOE-T2A

[0246] To allow for type II assembly (T2A) one-step cloning of sgRNAs and homology regions for DSB repair, the CRISPR / Cas9 plasmid pJOE8889.1 was modified as follows. 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 GoldenGate-based toolbox to create personalized integrative vectors for Bacillus subtilis. Sci. Rep. 7:14134) was modified to remove multiple restriction sites and the BpiI restriction site and ordered as a gene synthesis fragment (SEQ ID 005) flanked by SfiI restriction sites. The plasmid was 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 was ligated into SfiI-digested pJOE8999.1, followed by transformation into competent Escherichia coli DH10B cells. Positive clones were screened for purple colonies (blue-white screening and mRFP1 expression) on LB agar plates containing IPTG / X-Gal and kanamycin (20 μg / ml). The resulting plasmid with the verified sequence was named pJOE-T2A.

[0247] Plasmid pBW732

[0248] The 5' homology region (also called HomA) and 3' homology region (also called HomB) adjacent to the amylase amyB gene of Bacillus licheniformis DSM641 were ordered as synthetic gene synthesis fragments (SEQ ID006) flanked by XmaI restriction sites. Plasmid pJOE8999.1 and the synthetic amyB-HomAB fragment were digested with the restriction endonuclease XmaI, then ligated with T4-DNA ligase (NEB) and transformed into electrocompetent Escherichia coli DH10B cells. The correct plasmid was recovered and named pBW732.

[0249] Plasmid pBW742

[0250] Design 20 bp target sequences for the amyB gene for sgRNA using Geneious 11.1.5 (https: / / www.geneious.com). Anneal the resulting 5'-phosphorylated oligonucleotides SEQ ID 007 and SEQ ID 008 to form an oligonucleotide duplex. Construct a CRISPR / Cas9-based gene deletion plasmid for the amyB gene of Bacillus licheniformis using the following components: pBW732 and the oligonucleotide duplex (SEQ ID 007, SEQ ID 008) by type II assembly using the 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). Transform the reaction mixture into Escherichia coli DH10B cells (Life technologies). Spread the transformants on LB agar plates containing 20 μg / ml kanamycin and incubate overnight at 37 °C. Isolate plasmid DNA from independent clones and analyze the correctness by restriction digestion and sequencing. The resulting amyB deletion plasmid was named pBW742.

[0251] T2A CRISPR destination vectors pCC027 and pCC028

[0252] Plasmids pCC014 and pCC025 were modified such that the region covering the spacer-sgRNA and the amyB gene flanking the homology regions were replaced by the T2A cassette from plasmid pJOE-T2A. The backbones of pCC014 and pCC025 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, followed by PCR purification using the High Pure PCR Purification Kit, digestion with DpnI and gel purification. The corresponding backbone PCR fragments and T2A cassette PCR fragments were annealed in a 10 μl Gibson reaction and subsequently transformed into Escherichia coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37 °C. Plasmid DNA was isolated from independent clones and the correctness was analyzed by restriction digestion and sequencing. The resulting pCC014-derived and pCC025-derived T2A plasmid derivatives were named pCC027 and pCC028, respectively.

[0253] pCC029 - hag gene deletion plasmid

[0254] Design the 20bp target sequence of the hag gene for the sgRNA using Geneious 11.1.5 as previously described. Anneal the resulting 5'-phosphorylated oligonucleotides SEQ ID 056 and SEQ ID 057 to form the oligonucleotide duplex as described above. PCR amplify the genomic regions of the 5' and 3' of the hag gene on the genomic DNA of Bacillus licheniformis DSM641 using oligonucleotides SEQ ID 054 and Seq ID 053 and SEQ ID 052 and Seq ID 55, and then fuse them with the flanking oligonucleotides SEQ ID 053 and SEQ ID 054 by overlap extension PCR. Purify the resulting PCR product by column purification (Qiagen PCR purification kit). Construct the CRISPR / Cas9-based gene deletion plasmid for the hag gene of Bacillus licheniformis using the following components by type II assembly with the restriction endonuclease BsaI as described above: plasmid pCC027 (PV4-5 promoter variant), the fused homologous region of the hag gene with flanking BsaI restriction sites, and the oligonucleotide duplex (SEQ ID 056, SEQ ID 057). Transform the reaction mixture into Escherichia coli DH10B cells (Life technologies). Spread the transformants on LB agar plates containing 20 μg / ml kanamycin and incubate overnight at 37°C. Isolate plasmid DNA from independent clones and analyze the accuracy by restriction digestion and sequencing. The resulting hag gene deletion plasmid was named pCC029.

[0255] pCC030 - hag gene deletion plasmid

[0256] Construct the hag gene deletion construct as in pCC029, however using plasmid pCC028 (PV8-7 promoter variant).

[0257] pCC031–degU32 gene editing plasmid

[0258] Construct the degU32 genomic editing construct to introduce the degU H12L mutation as in pCC029, with the following modifications.

[0259] Order the gene synthesis construct (SEQ ID 058) with the degU H12L mutation introduced and a silent point mutation introduced to remove the PAM site in the degU32 homologous region as a gene synthesis construct with flanking BsaI sites (Geneart, Regensburg). Design the 20bp target sequence of the degU gene for the sgRNA as previously described and anneal the resulting 5'-phosphorylated oligonucleotides SEQ ID059 and SEQ ID 060 to form the oligonucleotide duplex.

[0260] pCC032 – degU32 gene editing plasmid

[0261] Generate the degU32 genome editing construct as described for pCC031, however using plasmid pCC028 (PV8-7 promoter variant).

[0262] pCC033 – amyE gene deletion plasmid

[0263] A fragment containing the amyE spacer - sgRNA and homology regions of the 5’ and 3’ regions of the Bacillus subtilis amyE gene was PCR amplified from plasmid pCC004 (WO17186550) using oligonucleotides SEQ ID 061 and SEQ ID 062 flanked by BsaI restriction sites. Subsequently, a CRISPR / Cas9-based gene deletion plasmid targeting the amylase amyE gene was constructed from plasmid pCC027 (PV4-5 promoter variant) and the PCR amplified fragment by type II assembly using the restriction endonuclease BsaI as described above. The reaction mixture was transformed into Escherichia coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37 °C. Plasmid DNA was isolated from independent clones and analyzed for correctness by restriction digestion and sequencing. The resulting amyE gene deletion plasmid was named pCC033.

[0264] pCC034 – amyE gene deletion plasmid

[0265] The amyE gene deletion construct was constructed as for pCC033, however using plasmid pCC028 (PV8-7 promoter variant).

[0266] pCC035 – aprE gene deletion plasmid

[0267] Order a fragment containing the aprE spacer (SEQ ID 064)-sgRNA and the homologous regions of the 5'- and 3'-regions of the Bacillus subtilis aprE gene as a synthetic gene fragment (SEQ ID 063) flanked by BsaI restriction sites. Subsequently, construct a CRISPR / Cas9-based gene deletion plasmid targeting the protease aprE gene with plasmid pCC027 (PV4-5 promoter variant) and the gene synthesis construct by type II assembly using the restriction endonuclease BsaI as described above. Transform the reaction mixture into Escherichia coli DH10B cells (Life technologies). Spread the transformants on LB agar plates containing 20 μg / ml kanamycin and incubate overnight at 37 °C. Isolate plasmid DNA from independent clones and analyze the correctness by restriction digestion and sequencing. The resulting aprE gene deletion plasmid is named pCC035.

[0268] pCC036 – aprE gene deletion plasmid

[0269] Construct the aprE gene deletion construct as in pCC035, however using plasmid pCC028 (PV8-7 promoter variant).

[0270] pCC037–pCC039–vpr gene deletion plasmid

[0271] Construct the CRISPR / Cas9 gene deletion constructs pCC037, pCC038, and pCC039 for the Bacillus licheniformis protease vpr gene as described for pCC035, however using a synthetic gene fragment (SEQ ID 065) containing the vpr spacer-sgRNA and the homologous regions of the 5'- and 3'-regions of the vpr gene. 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.

[0272] pCC040–pCC042–epr gene deletion plasmid

[0273] The CRISPR / Cas9 gene deletion constructs pCC040, pCC041, and pCC042 for the Bacillus licheniformis protease epr gene were constructed as described for pCC035, except that they were constructed using a synthetic gene fragment (SEQ ID 069) containing the epr spacer - sgRNA and the homology regions of the 5' and 3' regions of the epr gene. The resulting plasmids pCC040, pCC041, and pCC042 differ in the vpr spacer sequences (SEQ ID 070, SEQ ID 071, SEQ ID 072) within SEQ ID 069.

[0274] pCC043 – GFP gene integration plasmid

[0275] The 20 - bp target sequences of the Bacillus licheniformis amyB gene for the sgRNA were ordered as 5'-phosphorylated oligonucleotides SEQ ID 007 and Seq ID 008 and then annealed to form an oligonucleotide duplex. The 5' and 3' regions of the Bacillus licheniformis amyB gene were PCR - amplified using oligonucleotides SEQ ID 077 and SEQ ID 078 and SEQ ID 079 and SEQ ID 080, respectively.

[0276] A CRISPR / Cas9 - based gene integration plasmid construct for replacing the Bacillus licheniformis amyB gene was constructed by type II assembly using the restriction endonuclease BsaI as described above with the following components: pCC027, 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 region of the amyB gene. The reaction mixture was transformed into Escherichia coli DH10B cells (Lifetechnologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C. Plasmid DNA was isolated from independent clones and analyzed for correctness by restriction digestion and sequenced. The resulting CRISPR / Cas9 - based gene integration plasmid was named pCC043.

[0277] pCC044 – Bacillus pumilus sigE gene deletion plasmid

[0278] The CRISPR / Cas9 gene deletion construct pCC044 for the sigE gene of Bacillus pumilus DSM14395 was constructed as described for pCC035, except that it was constructed using a synthetic gene fragment (SEQ ID 082) containing the sigE spacer (SEQ ID 081)-sgRNA and the homology regions of the 5' and 3' regions of the sigE gene.

[0279] pCC045 - Bacillus pumilus sigF gene deletion plasmid

[0280] The CRISPR / Cas9 gene deletion construct pCC045 of the sigF gene of Bacillus pumilus DSM14395 was constructed as described for pCC035, however using a synthetic gene fragment (SEQ ID 084) containing the sigF spacer (SEQ ID 083)-sgRNA and the homology regions of the 5' and 3' regions of the sigF gene.

[0281] pCC046 - Bacillus pumilus spoIIE gene deletion plasmid

[0282] The CRISPR / Cas9 gene deletion construct pCC046 of the spoIIE gene of Bacillus pumilus DSM14395 was constructed as described for pCC035, however using a synthetic gene fragment (SEQ ID 086) containing the spoIIE spacer (SEQ ID 085)-sgRNA and the homology regions of the 5' and 3' regions of the spoIIE gene.

[0283] Example 1: Construction of a CRISPR / Cas9 genome editing plasmid carrying a constitutive promoter

[0284] To introduce a constitutive promoter driving the expression of the Cas9 enzyme in plasmid pBW742, a two-step method was employed.

[0285] First, 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.

[0286] By Gibson assembly HiFi DNA Assembly Cloning Kit, New England Biolabs) to integrate the terminator sequence t1t2t0 upstream of the mannitol promoter into pBW742. For this purpose, pMutin2 (accession number AF072806) was used as a template, and the terminator fragment (0.44 kb) was amplified by PCR with oligonucleotides SEQ ID 024 and SEQ ID 025. The corresponding vector backbone of pBW742 was amplified with oligonucleotides SEQ ID 022 and SEQ ID 023. The pBW742 amplicon was purified using a 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 at a 1:2 ratio at 50 °C for 1 hour. Escherichia coli strain DH10B was transformed with the assembly reaction and then plated on LB agar plates containing 20 μg / ml kanamycin. Plasmid DNA was isolated from independent clones and analyzed for correctness by restriction digestion and sequenced.

[0287] There are deviations from the published pMutin2 reference sequence. SEQ ID 026 covers a part of the pMutin2 sequence, and SEQ ID 027 covers the sequence deviations present in the corresponding region of pMutin2 present in the resulting plasmid pCC009.

[0288] Secondly, the mannitol-inducible promoter PmanP was replaced by two promoter variants of the Bacillus subtilis constitutive promoter Pveg – 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 contain the Pveg promoter, a standardized TSS (transcription start site) region, and a standardized ribosome-binding site region R0 derived from an adapted Pveg promoter library, where the promoter library was screened for altered expression levels relative to the promoter variants at the single-copy level in Bacillus subtilis. The promoter sequences PV4 and PV8 are listed as SEQ ID 028 and SEQ ID 029, respectively.

[0289] The integration of both promoter variants was carried out by Gibson assembly. The PV4 fragment and the PV8 fragment were amplified step by step. For the promoter fragments, pCC009 was used as a template, oligonucleotides SEQ ID 024 and SEQ ID 030 were used for the first PCR (Phusion High-Fidelity DNA Polymerase – NEB) and the resulting product served as a template for the second PCR, with oligonucleotides SEQ ID 024 and SEQ ID 031 for PV4 and SEQ ID 024 and SEQ ID 033 for PV8.

[0290] The vector backbone of pCC009 was PCR amplified using oligonucleotides SEQ ID 022 and SEQ ID 032. After purifying the vector amplicon with a PCR purification kit (Roche), the PCR product was digested with DpnI to remove the remaining circular plasmid DNA from the PCR reaction. Subsequently, the digested vector and the two promoter fragments were purified using a Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany). Subsequently, the vector amplicon pCC009 was annealed separately with the promoter fragments PV4 and PV8, thus replacing the mannose promoter PmanP with the PV4 variant and the PV8 variant of the Pveg promoter.

[0291] Subsequently, the annealing reaction was transformed into Escherichia coli DH10B cells (Life technologies). The transformants were spread on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37 °C. Plasmid DNA was isolated from 9 individual clones of the PV4 promoter and from 8 independent clones derived from the promoter variant PV8 and the correctness was analyzed by sequencing.

[0292] Table 1 summarizes the sequencing results of the various promoter variants:

[0293] Analysis of the clones from the PV4-cloning reaction revealed that only sequences with point mutations, nucleotide insertions or deletions within the PV4 region could be recovered.

[0294] Analysis of the clones from the PV8-cloning reaction revealed 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.

[0295] Table 1

[0296] 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

[0297] Gene deletion efficiency of the CRISPR / Cas9-based deletion plasmid

[0298] 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 (carrying different promoter variants as described in Table 1) isolated from Escherichia coli INV110 cells (Life technologies). Subsequently, they were plated on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37 °C.

[0299] The next day, 20 clones from each transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 009 and SEQ ID 010 to analyze the successful deletion of the amyB gene based on CRISPR / Cas9, and were further transferred to fresh LB agar plates without antibiotics and then incubated overnight at 48 °C for plasmid curing.

[0300] Based on the appearance of smaller specific PCR amplicons compared to the larger specific PCR amplicons of the wild-type amyB locus, the amyB gene deletion efficiency of each CRISPR / Cas9-based deletion plasmid was calculated as the percentage of successful gene deletions relative to the total number of analyzed clones.

[0301] As Figure 3 shown, the CRISPR / Cas9-based amyB gene deletion plasmids pCC010, pCC019, and pCC022 were not functional in Bacillus licheniformis like all the analyzed cells carrying the wild-type amyB locus.

[0302] Other promoter variants were functional in Bacillus licheniformis, driving Cas9 expression. In particular, the gene deletion plasmids pCC014, pCC016, pCC025 with promoter variants PV4-5, PV4-7, and PV8-7 respectively showed the highest gene deletion efficiency, greater than 60%.

[0303] The correct single clone was streaked onto a fresh LB agar plate without antibiotics and then incubated overnight at 48 °C for a second time for plasmid curing. The final clone was again analyzed for successful deletion of the amyB gene by colony PCR and for plasmid loss by plating on LB agar plates containing 20 μg / ml kanamycin. The resulting Bacillus licheniformis strain that had cured the deletion plasmid (sensitive to kanamycin) and deleted the amyB gene was named Bacillus licheniformis P310.

[0304] Example 2: Gene deletion and gene mutation using promoter PV4-5 and PV8-7 in Bacillus licheniformis

[0305] Electrocompetent Bacillus licheniformis P308 cells were prepared as described above and transformed with 1 μg each of the hag deletion plasmids pCC029 and pCC030 having promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046) respectively isolated from Escherichia coli INV110 cells (Life technologies), and subsequently plated on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C.

[0306] The next day, 20 clones from each transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 087 and SEQ ID 088 to analyze the successful deletion of the hag gene based on CRISPR / Cas9, and further transferred to fresh LB agar plates without antibiotics and then incubated overnight at 48°C for plasmid curing.

[0307] Based on the appearance of smaller specific PCR amplicons as expected compared to the wild-type hag locus, the hag gene deletion efficiency of each CRISPR / Cas9-based deletion plasmid was calculated as the ratio of successfully deleted genes as a percentage relative to the total number of analyzed clones. Three experiments were performed for each hag gene deletion plasmid. As Figure 4 shown in A, the CRISPR / Cas9-based gene deletion efficiencies of plasmids pCC029 and pCC030 were 95% and 100% respectively.

[0308] To analyze the point mutation introduction efficiency, Bacillus licheniformis P308 cells were transformed with two degU mutant plasmids pCC031 and pCC032 as described for the deletion of the hag gene, and the mutant plasmids again differed in the promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046) driving constitutive Cas9 expression. The transformed Bacillus licheniformis cells were plated on LB agar plates containing 20 μg / ml kanamycin and then incubated overnight at 30°C. The mutation efficiency introducing the H12L degU mutation was calculated as the ratio of the successfully mutated degU genes as a percentage relative to a total of 20 analyzed clones, based on the appearance of a degU-specific PCR-amplicon that could be cleaved with the restriction endonuclease PstI compared to the native degU-specific PCR-amplicon of the wild-type degU locus when using oligonucleotides SEQ ID 089 and SEQ ID 090. Three experiments were performed for each degU gene deletion plasmid. As Figure 4 shown in B, the CRISPR / Cas9-based mutation efficiencies of plasmids pCC031 and pCC032 were 19% and 24% respectively.

[0309] Example 3: Gene deletion using promoters PV4-5 and PV8-7 in Bacillus subtilis

[0310] Electrocompetent Bacillus subtilis ATCC6051a cells were prepared as described above and transformed with 1 μg each of the amyE deletion plasmids pCC033 and pCC034, which were isolated from Escherichia coli DH10B cells and had promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046), respectively. Subsequently, they were plated on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37 °C.

[0311] The next day, 20 clones from each transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 091 and SEQ ID 092 to analyze the successful deletion of the amyE gene based on CRISPR / Cas9, and were further transferred to fresh LB agar plates without antibiotics and then incubated overnight at 48 °C for plasmid curing.

[0312] Based on the appearance of a smaller specific PCR amplicon compared to the wild-type amyE locus, the amyE gene deletion efficiency of each CRISPR / Cas9-based deletion plasmid was calculated as the percentage of the total number of analyzed clones with successful gene deletion. Three experiments were performed for each hag gene deletion plasmid. As Figure 5 shown in A, the CRISPR / Cas9-based amyE gene deletion efficiencies of plasmids pCC033 and pCC034 inside Bacillus subtilis were 97% and 100%, respectively.

[0313] Similar to the method described for deleting the amyE gene, the gene deletion efficiencies of plasmids pCC035 and pCC036 for deleting the aprE gene in Bacillus subtilis, which were dependent on promoters PV4-5 (SEQ ID 037) and PV8-7 (SEQ ID 046), were analyzed. However, the cells were incubated on LB agar plates containing 20 μg / ml kanamycin and then transformed overnight at 30 °C. Gene deletion was analyzed again by colony PCR using oligonucleotides SED ID093 and SEQ ID 094, and the gene deletion efficiency was calculated as described above for three independent transformation reactions. As Figure 5 shown in B, the CRISPR / Cas9-based aprE gene deletion efficiencies of plasmids pCC035 and pCC036 inside Bacillus subtilis were 32% and 47%, respectively.

[0314] Example 4: Gene deletion using promoters PV4-5 and PV8-7 and different spacers in Bacillus licheniformis

[0315] 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, which were isolated from Escherichia coli Ec#098 cells and had the promoter PV4-5 (SEQ ID 037) and different vpr-specific spacer sequences (SEQ ID 066–068). Subsequently, they were plated on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C.

[0316] The next day, 20 clones from each transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 095 and SEQ ID 096 to analyze the successful deletion of the vpr gene based on CRISPR / Cas9, and were further transferred to fresh LB agar plates without antibiotics and then incubated overnight at 48°C for plasmid curing.

[0317] Based on the appearance of smaller specific PCR amplicons as expected compared to the wild-type vpr locus, the vpr gene deletion efficiency of each CRISPR / Cas9-based deletion plasmid was calculated as the ratio of successful gene deletion as a percentage relative to the total number of clones analyzed. As Figure 6 shown in A, the CRISPR / Cas9-based vpr gene deletion efficiencies of plasmids pCC037, pCC038, and pCC039 were 100%, 100%, and 84%, respectively.

[0318] The gene deletion efficiency of the epr gene in Bacillus licheniformis by plasmids pCC040, pCC041, and pCC042, which had the promoter PV4-5 (SEQ ID 037) and different epr-specific spacer sequences (SEQ ID 070–072), was determined as described for the vpr gene. However, oligonucleotides SEQ ID 097 and SEQ ID 098 were used for colony PCR-based gene deletion analysis. As Figure 6 shown in B, the CRISPR / Cas9-based epr gene deletion efficiencies of plasmids pCC040, pCC041, and pCC042 were 87.5%, 100%, and 100%, respectively.

[0319] Example 5: Gene Integration in Bacillus licheniformis Using Promoters PV4-5 and PV8-7

[0320] Electrocompetent Bacillus licheniformis Bli#005 cells were prepared as described above and transformed with 1 μg of the gene integration plasmid pCC043, which had the promoter PV4-5 (SEQ ID 037) and was isolated from Escherichia coli Ec#098 cells. Subsequently, they were plated on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37°C.

[0321] The next day, 20 clones of the transformation reaction were subjected to colony PCR using oligonucleotides SEQ ID 009 and SEQ ID 010 to analyze the successful integration of the CRISPR / Cas9-based PaprE-GFPmut2 expression cassette to replace the Bacillus licheniformis amyB gene, and were further transferred onto fresh LB agar plates without antibiotics and then incubated overnight at 48 °C for plasmid curing.

[0322] Based on the appearance of the expected specific PCR amplicons compared to the wild-type amyB locus, the gene integration efficiency of the CRISPR / Cas9-based gene integration plasmid pCC043 was calculated as the ratio of successful gene integration as a percentage relative to the total number of analyzed clones. The experiment was performed twice. As Figure 7 shown, the gene integration efficiency of plasmid pCC043 based on CRISPR / Cas9 into Bli#005 was 67%.

[0323] The gene integration efficiency of the PaprE-GFPmut2 expression cassette using plasmid pCC043 was determined in a manner similar to that of Bacillus licheniformis strain P308, showing an average gene integration efficiency of 72% in two independent transformation reactions, as Figure 7 described.

[0324] Example 6: Gene deletion using promoter PV4-5 in Bacillus pumilus

[0325] Electrocompetent Bacillus pumilus DSM14395 cells were prepared as described above and transformed with 1 μg of sporulation gene deletion plasmids pCC044 (sigE), pCC045 (sigF), and pCC046 (spoIIE) having the promoter PV4-5 (SEQ ID 037) driving the expression of the Cas9 endonuclease. The plasmid DNA was isolated from Escherichia coli DH10B cells and methylated in vitro as described above and then transformed. The transformed Bacillus pumilus cells were plated on LB agar plates containing 20 μg / ml kanamycin and incubated overnight at 37 °C.

[0326] The next day, 20 clones of each transformation reaction were subjected to colony PCR, using oligonucleotides SEQ ID 099 and SEQ ID 100 for sigE deletion analysis, oligonucleotides SEQ ID 101 and SEQ ID 102 for sigF deletion, and oligonucleotides SEQ ID 103 and SEQ ID 104 for spoIIE deletion analysis. The single colonies were further transferred onto fresh LB agar plates without antibiotics and then incubated overnight at 48 °C for plasmid curing.

[0327] Based on the appearance of smaller specific PCR amplicons than those of the wild-type locus, the gene deletion efficiency of plasmids pCC044, pCC045, and pCC046 in Bacillus pumilus was calculated as the ratio of successful gene deletions to the total number of analyzed clones, expressed as a percentage. As Figure 8 shown, the CRISPR / Cas9-based gene deletion efficiencies of plasmids pCC044, pCC045, and pCC046 inside Bacillus pumilus were 43%, 56%, and 50%, respectively. Sequence Listing <110> BASF SE <120> Shuttle vector for expression in Escherichia coli and Bacillus <130> 191608WO01 <160> 104 <170> According to Wipo Std 25 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of pE194 <400> 1 tatatacagc tggattcaca aaaaataggc ac 32 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of pE194 <400> 2 tatatacagc tggattatgt cttttgcgca gtc 33 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of functional mRFP cassette <400> 3 tatatggatc cgtaatcagg gtatcgaggc 30 <210> 4 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplified functional mRFP cassette <400> 4 tatatggatc cctcattagg cgggctacta a 31 <210> 5 <211> 1345 <212> DNA <213> Artificial sequence <220> <223> Functional fragment: T2A mRFP cassette <400> 5 ggaatagatc tggccaacga ggcctcgagg atccgatatc atgcatggcg cgccaccctg 60 agacgaccct gatgcaggtg accctgagac cttcgcgccc agctgtctag ggcggcggat 120 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt 180 tcgactgagc ctttcgtttt atttgatgcc tttaattaag taccttgtcg gataaagctg 240 tgttatatta tgtcttggtg ttaaatacac acgcttaacg atttatgcag agggtgctgc 300 aggcggcagt tctgtacaaa aatgacctaa gcggaggaaa aaaaccatta tattaggagg 360 aaataacatg gcctcttcag aggatgttat taaagaattt atgcggttta aggtgaggat 420 ggaaggctcg gtgaacggac atgagttcga aattgaggga gaaggtgaag gccgccctta 480 tgaaggtact cagacagcga aattgaaagt cacgaaaggc ggaccgctgc cgtttgcttg 540 ggacattctc tcacctcaat ttcaatatgg ctcaaaagcc tacgtaaaac acccggctga 600 catccctgat tacttaaagc tatccttccc ggagggcttt aaatgggaac gagttatgaa 660 ttttgaggac ggcggcgtcg ttactgtcac acaggattct tcccttcagg atggcgaatt 720 tatttacaaa gtaaaacttc gtggaactaa cttcccaagt gatggtcccg tgatgcaaaa 780 aaaaacaatg ggatgggaag catctacgga acgtatgtat ccggaggatg gagccttaaa 840 gggtgaaatc aaaatgcgcc tgaaacttaa agatggcgga cactatgacg cggaagttaa 900 aacaacatat atggctaaaa aaccagtcca actgccggga gcatataaga cggatataaa 960 gttggacatt accagccata atgaagatta cacgattgtg gaacagtatg agagagcaga 1020 gggcagacat agcacaggcg cgtaagaatt aatgaaaaat aagcggcagc ctgcttttcc 1080 atgcgggctg ccgcttatcg ggttattgtc gtgactggga aaaccctggc gactagtctt 1140 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg 1200 ctcggttgcc gccgggcgtt ttttattggt gagaatccag gggtccccaa taattacgat 1260 ttggtctcac tcacacctgc tcgtctcact caatttaaat ggcggccgcg gatcctcgac 1320 gggccaataa ggccagatct ggatt 1345 <210> 6 <211> 1012 <212> DNA <213> Artificial sequence <220> <223> Homologous region: The 5' primer region and 3' primer region of the amyB gene are fused with the flanking XmaI sites <400> 6 cccgggataa tgccgtcgca ctggccgata ttgagagatt tccttgtgac aagctgcaaa 60 gcataatgat gacggtccag ctcgcggctg attcccgtta acagattcat ataataaggt 120 tctgttgtat ccatttcttc cagtatgagc agcttgacga cctgtgttct gttttgaacg 180 agcgctcttg ctgcatagtt cggtatataa ttgagctcct tcattgcgga atgaacaagc 240 tttttcaatt catccgtcac agtctcagga tgattgatca cccgcgatac cgtcattttc 300 gacacatttg ctttctttgc tacatcagat aacgttgcca tttcatcccc gccttaccta 360 tgcgattcaa actgtcagca agtccttcct gagggctgat gacactttgt taaaattaat 420 tataaaatgt aatcaaagaa atttataaga cgggcaaaat aaaaaaacgg atttccttca 480 ggaaatccgt cctctctgct cttctagatt ctcctcccct ttcaatgtga aacatatgat 540 attgtataaa tattccgaat ttttaacaaa taccattttc cctatatttt cttccaaaag 600 aaaagcgccg atatggcgct ttctactcat ttattcaata gcctctctgc ttcttcactt 660 cttcaagctg agatacagtt accaattgat agccttttgc tttcagcttt ttaataatct 720 cttcagcagc atctgcggac gttgcataaa tatcgtgcat taagacgatt tttccgtctc 780 ccgcatggct catgacatga ttgacaatct tttgcttatt tttgtacttc caatcttccg 840 gatcaacatc ccacaatgaa accttcagat tggaaagcga gcggacggaa tcattgatcc 900 cgccgtatgg aggacgcaag tgtacaggca ggtgtccgct gattttttcg atcatttctt 960 gcgtgtcgtt aatctcctga tacgcttttt cgtttgacag ccttgtcccg gg 1012 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: protospacer target sequence for the amyB gene with a 4nt 5 - primer extension part <400> 7 tacgtcgcag cagaaattaa gaga 24 <210> 8 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: protospacer target sequence for the amyB gene with a 4nt 5 - primer extension part <400> 8 aaactctctt aatttctgct gcga 24 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of the amyB genomic region <400> 9 ttgcccgaat acaacgacag 20 <210> 10 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of the amyB genomic region <400> 10 caacaacagg ctgtctgacg g 21 <210> 11 <211> 2143 <212> DNA <213> Bacillus licheniformis <220> <223> P300 DSM641 RMS: RMS region and flanking regions <400> 11 aacttctata aatgtaacga tacgatttat tgtttcatta aagtcttcct ttatttcatg 60 ttcccatatt cttttaatgt tccaatcctt ttcctcgtaa tatttattaa cttccttatc 120 tcttttttta tttctttcga gttttttctc ccaatattcc gtattacttt ttggtatatt 180 cccgtgtttt tcacacgcat gccagaaaca agaatcaatg aatatgacta ttttatattt 240 ctgtattact atatctggac taccgtataa tttcttaaca ttttttcgga atcttattcc 300 acggtgccat agttctttag taaccttatc ttctaatttt gaacgagatt tgattgcctg 360 catgtttttt cttctttgtt cttttgaaac cgtgtcagtc atagaagagt cctccaaagc 420 cacaataatt gtattctata aacgaggaag caagccctca agcttacccc ctcttagttc 480 cttttttgcc tacttattta tttgttttca ttttcaaatt catcataaga acccatttca 540 caaaaatcaa tggagtaatg ttgttcgctg tgtttataaa caattactga gtcaatattt 600 agtctttcca gcatttcata ggttagaagt tctttttcct ctaagttcat aacttgtctt 660 agtagccatt ctccaagagc actatttgga ttagacataa gtgctttact attgtcttgg 720 cataccttgg ctgataaaag cgatttgtct ggcaagcgta actgaaaagg tttatcacga 780 gctgggaaaa atgttgggaa tacattatga atccattttg gaattggtat ataaatctcg 840 ttagggtttc gtggtcgacc taaagcattc cattggttta gaccgctttt ttctggtaca 900 tgacgctttg agccacggtc tgaaaagagt ggaagaataa cgtgctcaag gttttcaaaa 960 ggattgacag ttggtgctgg aatttttgga atttcaaagc caaatagttt agccaattca 1020 tgataaggat tttctaagat ttcaacatta atttcttcaa taggtttatc agtgataaaa 1080 cgcttataaa gggtgctctt agtgacatta aagctgtatt cgtgtagacc gtcttcaaag 1140 gtgattgtat ttctgttgtt acttactttc acatttgtaa ttgaggagat ttcaaccaag 1200 tccattggct cttcaaaaat aagaattttc cctggctttc ttgttacaca gtggtatatc 1260 attgaatcaa taccatatgt tcttttagta aattcaattc tctcgttacg gagagaagca 1320 accgtgttta ttagctcttt tggagatttc ccacgataca agtctgagtc tttattgaat 1380 tcagctactt tttgaagagt atgaccatta ccatgaagaa aagtcttaat accaattccg 1440 acacgattta atgaagcgtc agcagaacag tctgacctcc ccaagttttc agctccaaat 1500 gcttcacaaa aagcattttc cacattcctt gagaccaaat aaggcgagtc actttcagag 1560 aacaaattgg atagcgaacc agttgagcgg agcatttgtt tgtatgtagt gcagttgatg 1620 gctggttgat tagtatagaa cattattttt cctcctcttt tatgcttgtc atttcttctt 1680 tcagacccaa aaggtagtca gctgatacgt tcaatgtttc agctattctt ttgaaagtgt 1740 ccaatgatgg agttctattt tcactttcat atagtgacca agtgcttcta gtgaccccga 1800 ctttttcagc gatttggctg ggtaataacc tacgagcttc tcttgcattt tgaatacgat 1860 ttccaaggaa aggtatcatt tttgcacctc caagatttgt tgttttcaga gtatcaccag 1920 aacccccgaa aatagtccaa agttagctaa cagcaaacaa ataaaaataa ataagttgtt 1980 tactcttagc aaacttgtta ctaaaatttg ataaagttat tcatttaatc cagctcttat 2040 gctaaaattg cattagcgga caagcttaat gtttgcaagg aggtataatt ttgacttatc 2100 gagtaggtag tatgtttgct gggataggtg gaacttgttt agg 2143 <210> 12 <211> 1146 <212> DNA <213> Bacillus licheniformis <220> <223> Coding region: Restriction enzyme P300 DSM641 <400> 12 ttatttgttt tcattttcaa attcatcata agaacccatt tcacaaaaat caatggagta 60 atgttgttcg ctgtgtttat aaacaattac tgagtcaata tttagtcttt ccagcatttc 120 ataggttaga agttcttttt cctctaagtt cataacttgt cttagtagcc attctccaag 180 agcactattt ggattagaca taagtgcttt actattgtct tggcatacct tggctgataa 240 aagcgatttg tctggcaagc gtaactgaaa aggtttatca cgagctggga aaaatgttgg 300 gaatacatta tgaatccatt ttggaattgg tatataaatc tcgttagggt ttcgtggtcg 360 acctaaagca ttccattggt ttagaccgct tttttctggt acatgacgct ttgagccacg 420 gtctgaaaag agtggaagaa taacgtgctc aaggttttca aaaggattga cagttggtgc 480 tggaattttt ggaatttcaa agccaaatag tttagccaat tcatgataag gattttctaa 540 gatttcaaca ttaatttctt caataggttt atcagtgata aaacgcttat aaagggtgct 600 cttagtgaca ttaaagctgt attcgtgtag accgtcttca aaggtgattg tatttctgtt 660 gttacttact ttcacatttg taattgagga gatttcaacc aagtccattg gctcttcaaa 720 aataagaatt ttccctggct ttcttgttac acagtggtat atcattgaat caataccata 780 tgttctttta gtaaattcaa ttctctcgtt acggagagaa gcaaccgtgt ttattagctc 840 ttttggagat ttcccacgat acaagtctga gtctttattg aattcagcta ctttttgaag 900 agtatgacca ttaccatgaa gaaaagtctt aataccaatt ccgacacgat ttaatgaagc 960 gtcagcagaa cagtctgacc tccccaagtt ttcagctcca aatgcttcac aaaaagcatt 1020 ttccacattc cttgagacca aataaggcga gtcactttca gagaacaaat tggatagcga 1080 accagttgag cggagcattt gtttgtatgt agtgcagttg atggctggtt gattagtata 1140 gaacat 1146 <210> 13 <211> 1022 <212> DNA <213> Artificial Sequence <220> <223> Homology Region: The 5' and 3' primer regions of the restriction enzyme gene are fused with the flanking BsaI sites <400> 13 ggtctcgacc caacttctat aaatgtaacg atacgattta ttgtttcatt aaagtcttcc 60 tttatttcat gttcccatat tcttttaatg ttccaatcct tttcctcgta atatttatta 120 acttccttat ctcttttttt atttctttcg agttttttct cccaatattc cgtattactt 180 tttggtatat tcccgtgttt ttcacacgca tgccagaaac aagaatcaat gaatatgact 240 attttatatt tctgtattac tatatctgga ctaccgtata atttcttaac attttttcgg 300 aatcttattc cacggtgcca tagttcttta gtaaccttat cttctaattt tgaacgagat 360 ttgattgcct gcatgttttt tcttctttgt tcttttgaaa ccgtgtcagt catagaagag 420 tcctccaaag ccacaataat tgtattctat aaacgaggaa gcaagccctc aagcttaccc 480 cctcttagtt ccttttttgc ctacttattt atatttttcc tcctctttta tgcttgtcat 540 ttcttctttc agacccaaaa ggtagtcagc tgatacgttc aatgtttcag ctattctttt 600 gaaagtgtcc aatgatggag ttctattttc actttcatat agtgaccaag tgcttctagt 660 gaccccgact ttttcagcga tttggctggg taataaccta cgagcttctc ttgcattttg 720 aatacgattt ccaaggaaag gtatcatttt tgcacctcca agatttgttg ttttcagagt 780 atcaccagaa cccccgaaaa tagtccaaag ttagctaaca gcaaacaaat aaaaataaat 840 aagttgttta ctcttagcaa acttgttact aaaatttgat aaagttattc atttaatcca 900 gctcttatgc taaaattgca ttagcggaca agcttaatgt ttgcaaggag gtataatttt 960 gacttatcga gtaggtagta tgtttgctgg gataggtgga acttgtttag gctcaggaga 1020 cc 1022 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplified restriction enzyme genomic region <400> 14 gacaatcccc ttttactgac c 21 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplified restriction enzyme genomic region <400> 15 ctatttcatt tgcccagaca atcc 24 <210> 16 <211> 1363 <212> DNA <213> Artificial Sequence <220> <223> Homologous region: 5 - primer region and 3 - primer region of pga gene fused with flanking BsaI sites <400> 16 ggtctcgacc cgaacactga aattttagac cgggctggga tatcgagaca tatatagggg 60 cgtttaatgg cgaatacagt aacaatgaga atagtaagaa aaattaaaaa tgttaaagtt 120 tgatgaatta tcattgaaaa aaattaatgg ctttttaaat cctaggattt taacctaaaa 180 tctgaagaaa taaggtggat cgaacgactc acaaaatatt tggatttgtc aatgaatccc 240 gctttatgct aaaagagatt ttcatttttt gatagatggt ctgattgtca taggacggat 300 ttgttttgaa gagggaacat tggtgacttt ttaacctgtt cgaaaagagc gaaaatacta 360 aaagaaaaga gacatcccgg ctgacagccc atttaaaggg gattgcggcc gggggaaaaa 420 agagatcctg aatccatcct tcaacctttc atctgaaata gggagaaaag tacaaaaatc 480 ataatgtcga attttgaaag cgcatactta aaacgctgac aaaaatctga taggaattaa 540 gaactttcga tttccaaaaa tatcaataaa aagataggca ttaatgactc gggcgaggtg 600 atctttgtca cggaaaattt cgtcgtcttc tgttacataa tgccgattgt gatttcatag 660 tgaaccctga tcccggttat aaaagacctg tgaaaagcgg ccggtttgaa agggaaacac 720 gacaattttc ttaaccggtc agtgtataaa gttttataga aaatcaggag gatatataca 780 tggttttggg gttcatgttt attgtattct tttgaaggga ataaaaactg acaaatttcg 840 actgaagcaa aatttgaaaa tgcatcacct taccaattcg ggatgggaac cgcacctcat 900 gttcatgacc tctttagaat atttcccttc atctttttaa tccgcgctta ggtgaaaaag 960 ctgatcatgc tgtgctgagc gtttcttctc gctatgacgc tgctgtacat gcaaaaaaag 1020 tcctttaaat atcccagttg aatgacgatg aaagaggaaa gaagaggagg aacagatcaa 1080 ttgataaaaa aagcggcaaa caaaaagttg gttttgtttt gtggaattgc ggtgctttgg 1140 atgtctttat ttttaacgaa tcataatgat gtacgcgccg atacgatcgg cgagaaaata 1200 gcggaaactg ccagacagct tgagggtgcg aaatacagct acggcggaga gaagccgaaa 1260 acggggtttg actcgtcagg ctttgtgcaa tatgtgtttc aatcgctcga tattacgctt 1320 ccgagaacgg taaaggaaca atcgactctt ggctcaggag acc 1363 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of the pga genomic region <400> 17 aaagccttct cctctctatt 20 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of the pga genomic region <400> 18 ttcttgaaaa agacaaggtc 20 <210> 19 <211> 1027 <212> DNA <213> Artificial sequence <220> <223> Homology region: 5' primer region and 3' primer region of the aprE gene fused with flanking BsaI sites <400> 19 ggtctcgacc cgaagttctt ttttaacata taggtaaaac aatacgaaaa aaggcgccaa 60 gtattgaaga attgcagcag ccgcggcatt tcccttttcg attgaagcaa aaaacgtata 120 ttgaacagta agcattccaa aaatggaaaa tactaaaatc gaacaaatat ctgttttttt 180 cttccatatc tgacacacat gttgaaaacc gtttttcatt gaaacatata acaagagaat 240 gactcccgat gccagaagcc tgacagagac aagcgagccg gcttcaaccg ctcccctttc 300 aaatatgtac tgtgcagcgc ttcccgataa tccccacaat gaagcccctg caagcaccat 360 caatacgcct ttcacatgag ctgatttcat atctttcacc cgtttctgta tgcgatatat 420 tgcatatttt aatagatgat cgacaaggcc gcaacctcct tcggcaaaaa atgatctcat 480 aaaataaatg aatagtattt tcataaaatg agctcaataa catattctaa caaatagcat 540 atagaaaaag ctagtgtttt tagcactagc tttttcttca ttctgatgaa ggttgttcaa 600 tattttgaat ccgttccatg atcgtcggat ggccgtattt aaaaatcttg acgagaaacg 660 gcgggtttgc ctcgctcagc ccggcttttg agagctcttg aaacgtcgaa accgctgcat 720 cgctgttttg cgtcagttca atcgcatact ggtcagcagc tttttcctga tgcctcgaaa 780 ctgcgttcgt aaatggagac gacgcgaaag agatgacccc catcagcatc agaagaagcg 840 gaagtgcggc tagatcggat tttcctgcaa tatgaaggct tcttccatag cggccgatga 900 tccgcttgta cagcttgtcg atcacataaa agacagcaag ggataaaagc agatacccgc 960 caagtcctat gtaaacatgc ttcatcacat agtgccccat ttcgtgcgcc atgatgctca 1020 ggagacc 1027 <210> 20 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of the aprE genomic region <400> 20 ccggttgtca ttgatccttt a 21 <210> 21 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of the aprE genomic region <400> 21 atcctcctgc aaaaaccgta t 21 <210> 22 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of pBW742 <400> 22 caaatttaca aaagcgactc gtgagttttc gttccactga g 41 <210> 23 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of pBW742 <400> 23 gaacgttgct ctagagttaa gggattttgg tcatgg 36 <210> 24 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of the terminator region of pMutin2 <400> 24 gtggaacgaa aactcacgag tcgcttttgt aaatttgg 38 <210> 25 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: Terminator region of pMutin2 amplified by PCR <400> 25 catgaccaaa atcccttaac tctagagcaa cgttcttgcc 40 <210> 26 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Functional fragment: Terminator region of pMutin2 <400> 26 ggggatctct gcagtgagat ct 22 <210> 27 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Sequence of the terminator region in pCC009 <400> 27 ggggatctct gcagtcggga agat 24 <210> 28 <211> 128 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 28 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttggagaa taaatgtgga gaaagattaa ctaataagga 120 ggacaaac 128 <210> 29 <211> 128 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 29 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttgaagaa taaatgtgga gaaagattaa ctaataagga 120 ggacaaac 128 <210> 30 <211> 101 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 30 aacacgagcc catttttgtc aaataaaatt taaccggtat caacgttaat aagacgttgt 60 caataaaatt attttgacaa aattttaata atccaaatga g 101 <210> 31 <211> 87 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 31 ctattgagta tttcttatcc atgtttgtcc tccttattag ttaatctttc tccacattta 60 ttctccaaca cgagcccatt tttgtca 87 <210> 32 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 32 gattaactaa taaggaggac aaacatggat aagaaatact caataggc 48 <210> 33 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 33 ctattgagta tttcttatcc atgtttgtcc tccttattag ttaatctttc tccacattta 60 ttcttcaaca cgagcccatt tttgtca 87 <210> 34 <211> 127 <212> DNA <213> Artificial Sequence <220> <223> Promoter Variant <400> 34 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa atgggctcgt gttggagaat aaatgtggag aaagattaac taataaggag 120 gacaaac 127 <210> 35 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> Promoter Variant <400> 35 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttggagaa aatgtggaga aagattaact aataaggagg 120 acaaac 126 <210> 36 <211> 127 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 36 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttgggaat aaatgtggag aaagattaac taataaggag 120 gacaaac 127 <210> 37 <211> 129 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 37 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgaacaa aaatgggctc gtgttggaga ataaatgtgg agaaagatta actaataagg 120 aggacaaac 129 <210> 38 <211> 127 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 38 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttgagaat aaatgtggag aaagattaac taataaggag 120 gacaaac 127 <210> 39 <211> 127 <212> DNA <213> Artificial Sequence <220> <223> Promoter Variant <400> 39 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttggaaat aaatgtggag aaagattaac taataaggag 120 gacaaac 127 <210> 40 <211> 127 <212> DNA <213> Artificial Sequence <220> <223> Promoter Variant <400> 40 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aagggctcgt gttggagaat aaatgtggag aaagattaac taataaggag 120 gacaaac 127 <210> 41 <211> 127 <212> DNA <213> Artificial Sequence <220> <223> Promoter Variant <400> 41 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttgaagaa aaatgtggag aaagattaac taataaggag 120 gacaaac 127 <210> 42 <211> 130 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 42 aattttgtca aaataatttt attgacaacg tcttattaac cgttgatacc ggttaaattt 60 tatttgacaa aaatgggctc gtgttgaaga ataaatgtgg agaaagatta actaataagg 120 gaggacaaac 130 <210> 43 <211> 127 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 43 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttgaagaa taaatgtgga aaagattaac taataaggag 120 gacaaac 127 <210> 44 <211> 128 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 44 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccc ggttaaattt 60 tatttgacaa aaatgggctc gtgtgaagaa taaatgtgga gaaagattaa ctaataagga 120 ggacaaac 128 <210> 45 <211> 127 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 45 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa aatgggctcg tgttgagaat aaatgtggag aaagattaac taataaggag 120 gacaaac 127 <210> 46 <211> 127 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 46 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 atttgacaaa atgggctcgt gttgaagaat aaatgtggag aaagattaac taataaggag 120 gacaaac 127 <210> 47 <211> 129 <212> DNA <213> Artificial sequence <220> <223> Promoter variant <400> 47 aattttgtca aaataatttt attgacaacg tcttattaac gttgataccg gttaaatttt 60 attttgacaa aaatgggctc gtgttgaaga ataaatgtgg agaaagatta actaataagg 120 aggacaaac 129 <210> 48 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 48 gaagttttag atgccactct tatccatcaa tccatcactg 40 <210> 49 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 49 catctcaaat ttcgcattta ttccaatttc ctttttgcgt g 41 <210> 50 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 50 cacacgcaaa aaggaaattg gaataaatgc gaaatttgag 40 <210> 51 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 51 gaccagtgat ggattgatgg ataagagtgg catctaaaac 40 <210> 52 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 52 caagctatgc ttgctcaagc 20 <210> 53 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 53 ctgttggttc gcttgagcaa gcatagcttg gacggttcag cgtgttaagc 50 <210> 54 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 54 ggtggtggtc tctaccccga tcgaagagcc attcgag 37 <210> 55 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 55 ggtggtggtc tcttgagctt cctctgtccg attgtcc 37 <210> 56 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 56 tacggcaatc tctgaaaaaa tgag 24 <210> 57 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 57 aaacctcatt ttttcagaga ttgc 24 <210> 58 <211> 814 <212> DNA <213> Artificial sequence <220> <223> Homology region: Homology region of mutant degU gene with flanking BsaI sites <400> 58 ggtctcgacc cgatttggga ttgataccga cgctcagaaa atacttgaac acgatcgaag 60 attatcatgg aaaagcaaag attcatttcc aatgcatcgg agaatccgaa gaaagaagaa 120 tagcaccgcg gtttgaggtt gcactattcc ggcttgcaca ggaagcggtg acaaacgcct 180 taaaacactc cgaatcaact gaaattcatg ttaaagtaga agtgacaaaa gattttgtga 240 cgctgattat caaagacaat ggaaacggct ttgacttaaa agaagtaaaa ggcaagaaga 300 acaaatcttt cggtctgcta ggtatgaaag aaagagtcga tttgctcgaa ggctcaatga 360 caatcgattc gaaaataggt cttgggacat ttatattgat taaagttcca ctgtctttgt 420 aaagataatt gtaaaataga gacaaaagac atattgacca taaaagcggt gtgtttaaca 480 atgagaatgg ggaggcgtag cttgtgacta aagtaaatat tgtaattatt gacgatctgc 540 agttattccg tgaaggtgtc aaacggattt tggatttcga gcctaccttt gaagtagtgg 600 ccgaaggaga cgacggagat gaagcggctc gcattgtcga gcactaccat cctgatgttg 660 ttatcatgga tattaatatg ccgaatgtga acggagtaga agcgacaaaa caactggtcg 720 acttgtatcc ggaatcaaag gttattattt tatccatcca tgatgacgaa aactatgtta 780 cacatgcatt aaaaacagga gccctcagga gacc 814 <210> 59 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 59 tacgggattt cgaacctacc tttg 24 <210> 60 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 60 aaaccaaagg taggttcgaa atcc 24 <210> 61 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 61 ggttgcggtc tcatacgtga agatcaggct atcactggt 39 <210> 62 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide <400> 62 caacgggtct cttgagttgg caggccgctg aatttc 36 <210> 63 <211> 1435 <212> DNA <213> Artificial sequence <220> <223> sgRNA and homology region: protospacer aprE-sgRNA, Homology region of aprE gene with flanking BsaI sites <400> 63 ggttgcggtc tcatacggaa acaaacccat accaggagtt ttagagctag aaatagcaag 60 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaagcttag 180 gcccagtcga aagactgggc ctttttaata cgactcacta tagggtcgac ggccaacgag 240 gcccattttc ttctgctatc aaaataacag actcgtgatt ttccaaacga gctttcaaaa 300 aagcctctgc cccttgcaaa tcggatgcct gtctataaaa ttcccgatat tggttaaaca 360 gcggcgcaat ggcggccgca tctgatgtct ttgcttggcg aatgttcatc ttatttcttc 420 ctccctctca ataatttttt cattctatcc cttttctgta aagtttattt ttcagaatac 480 ttttatcatc atgctttgaa aaaatatcac gataatatcc attgttctca cggaagcaca 540 cgcaggtcat ttgaacgaat tttttcgaca ggaatttgcc gggactcagg agcatttaac 600 ctaaaaaagc atgacatttc agcataatga acatttactc atgtctattt tcgttctttt 660 ctgtatgaaa atagttattt cgagtctcta cggaaatagc gagagatgat atacctaaat 720 agagataaaa tcatctcaaa aaaatgggtc tactaaaata ttattccatc tattacaata 780 aattcacaga atagtctttt aagtaagtct actctgaatt tttttaaaag gagagggtaa 840 agataatagt aaaaagaagc aggttcctcc atacctgctt ctttttattt gtcagcatcc 900 tgatgttccg gcgcattctc ttctttctcc gcatgttgaa tccgttccat gatcgacgga 960 tggctgcctc tgaaaatctt cacaagcacc ggaggatcaa cctggctcag ccccgtcacg 1020 gccaaatcct gaaacgtttt aacagcggct tctctgttct ctgtcaactc gatcccatac 1080 tggtcagcct tattctcctg ataacgcgag acagcattag aaaaaggcgt aaccgcaaag 1140 ctcaaaacag aaaacaaaag caataacagc ggaagtgccg caagatcatg ccgcccttct 1200 aaatgaaaca tgctgcgggt taggcgaacc gtccgcttgt aaagcttatc aatgacataa 1260 aatccggcga gcgacacgag caaatagcca gccagaccga tgtaaacgtg cttcatgaca 1320 taatggccca tttcgtggcc cataataaac agaatttctg aatcgtcaag tttgttcagc 1380 gtcgtatccc acaatacaat ccgtttattg gccccaattc tcaagagacc cgttg 1435 <210> 64 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Protospacer: Protospacer target sequence for the aprE gene <400> 64 gaaacaaacc cataccagga 20 <210> 65 <211> 1241 <212> DNA <213> Artificial sequence <220> <223> sgRNA and homology region: Protospacer vpr-sgRNA, vpr gene homology region with flanking BsaI sites <400> 65 ggttgcggtc tcatacgatc aggaacggaa cgagtcggtt ttagagctag aaatagcaag 60 ggttgcggtc tcatacgatc aggaacggaa cgagtcggtt ttagagctag aaatagcaag 60 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180 cagtcgatac ggatccacta gtctatctct tctctttttt ccgaaaagcc gcctgcctga 240 cagtcgatac ggatccacta gtctatctct tctctttttt ccgaaaagcc gcctgcctga 240 taagcatcgg cagcctgata tgtaccgccg gcgaacgccg tcaccttcat ctgattttct 300 taagcatcgg cagcctgata tgtaccgccg gcgaacgccg tcaccttcat ctgattttct 300 ctgacaggca tcaccgcatt cagcagcacg gctgtccata attttaaaaa tgatatcaaa 360 ctgacaggca tcaccgcatt cagcagcacg gctgtccata attttaaaaa tgatatcaaa 360 cctttcatac cgatccctcc agtttcgttt tgataaaact agcaactcta ttaaactttc 420 cctttcatac cgatccctcc agtttcgttt tgataaaact agcaactcta ttaaactttc 420 ttgctctatc ttatcccagc aaaatgaaaa tgtttgtcac aatgtgtgtg caaaatgatt 480 ttgctctatc ttatcccagc aaaatgaaaa tgtttgtcac aatgtgtgtg caaaatgatt 480 ctagttttta gaagttttgt tgaaaactga aggaatcgca tgattcagcg gatacaaacc 540 ctagttttta gaagttttgt tgaaaactga aggaatcgca tgattcagcg gatacaaacc 540 atgaatgtaa cttactcaca gcttatccta aggataaaca catattaccc acaggatata 600 atgaatgtaa cttactcaca gcttatccta aggataaaca catattaccc acaggatata 600 tccacatatc cacatactta ttcaatattt agtataagaa cgtatattcc ctacaatatc 660 tccacatatc cacatactta ttcaatattt agtataagaa cgtatattcc ctacaatatc 660 tatacacaag tttattcact tatacacagt aaattgtgca taaatctaat gacaagcctt 720 tatacacaag tttattcact tatacacagt aaattgtgca taaatctaat gacaagcctt 720 gttgagaacc actcaacaag gcttttttat gttaaaatac ggataatgcg ttcaggagaa 780 gctccccttc tcttcaaaac gtgaaaaaag caatcggagg acatcgtgta tatgctttct 840 tttatcgtat tattcggctt atccttcatt attgtctgct ttatattttt cacgactttg 900 tacttcgccg tcaacctgca gaagcgcgag cccaagcctt ttcaaaaagc tgcggagcaa 960 accgtcgata ccatcatcct cattccgctc agctggctgt ttaccgcttt atacatatgc 1020 attctgttta ttcttttccc aatccgccat tttctcgatt tttttcagca aaaacgctaa 1080 attgactgat gaaacgcttc ggccagcagc cggtatgaat ccaatctgtc ttgaaaatcg 1140 tgggtgatcg tcaccgccat gatttcgtcc gttccgtaag cgccggccag ttcaagcagc 1200 tgttcctagc tcgagccatg gctcactcaa gagacccgtt g 1241 <210> 66 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Protospacer: Protospacer target sequence for the vpr gene <400> 66 atcaggaacg gaacgagtcg 20 <210> 67 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Original spacer region: The original spacer target sequence for the vpr gene <400> 67 gcttccgtat aatgagtatt 20 <210> 68 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Original spacer region: The original spacer target sequence for the vpr gene <400> 68 cacgatccga aaaacccgta 20 <210> 69 <211> 1338 <212> DNA <213> Artificial sequence <220> <223> sgRNA and homology region: The epr-sgRNA of the original spacer region, The homology region of the epr gene with flanking BsaI sites <400> 69 ggttgcggtc tcatacgatt ccggtccagc gcttttagtt ttagagctag aaatagcaag 60 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180 ggcccagtcg atacgtaaag actgggcctt tttaatacga ctcactatag ggtcgacggc 240 caacgaggcc tcgaggatcc gatatcatgc atggcgcgcc accctgagac gaccctgatg 300 caggtgaccc ggatccacta gtccgatttg acatcgtgct gtttaaagga cctgacaaag 360 atatattcat taaaagggtg atcgggcttc cgggcgaaac cctcaggtat gaagatgatc 420 agctgtatat caacgaagaa aagatcaaag agccttatct ggacgactta aaggccgtca 480 ccgccggagg ggacttgaca ggggatttta cactgcagga agtgaccgga gaggagaagg 540 tgcctgaaaa cgagtacttc gtcctcgggg acaaccggat ccacagcttt gacagccgcc 600 atttcggctt tgtttcagaa cgggacatcg tcgggattgt gacggaaaga attgataaga 660 agtgattgga gagtacgggg gagagtaagc ggccgaccaa ggaatacgat tacgcaaatg 720 acgagcccga aatgtcaatt agtacaacag catcaataat gacgcatttg ctaaatatga 780 aaattaaaag gcccggatga ttccgggctt ttttccgtac taagcggcgt tcgctatata 840 tatcggagga tttttgaatt ttcaaagaga aagaaagctt atcttaaggt cgcttgtcat 900 gcacctttag tttttaaaac gttaaaaaca ctgttatatc aacatttgtg aagcttcctg 960 tttattcggg aagttaaatt gggtactcca agttagtttt aaaaaagagt cataaggcca 1020 gcttctatcg atgaatcatt tttaagcgac gccttttgtc taaaatgtat aatgttactt 1080 ttgtttttgt tgaaagtgaa caatgttatt gactggctta caacctacaa tttaattaaa 1140 taaaaaatag attaaaagaa gggagcgttc tcataccgtg gaaaaaacaa ttaaacatga 1200 ccccaattat tacaaaaaga taattattgc attgtgttta gggtgggtcg ctatttggat 1260 ttatcgtaca atacttacgc caatatatcc gcagattcaa gaatcattag ggaatattag 1320 tgctcaagag acccgttg 1338 <210> 70 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Protospacer: Protospacer target sequence for the epr gene <400> 70 attccggtcc agcgctttta 20 <210> 71 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Protospacer: Protospacer target sequence for the epr gene <400> 71 cgctttttca gctttggcaa 20 <210> 72 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Protospacer: Protospacer target sequence for the epr gene <400> 72 gcaaacatgc cggtgacgtg 20 <210> 73 <211> 865 <212> DNA <213> Artificial Sequence <220> <223> Functional fragment: T2A lacZ cassette <400> 73 gcgatgttaa ggaccgtctc atctcacctg caaggtctca tctcttttac tccatctgga 60 tttgttcaga acgctcggtt gccgccgggc gttttttatc taaaactagt gtcgagggtc 120 ttcggtaccg cgatttacat atgctggcac gacaggtttc ccgactggaa agcgggcagt 180 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt 240 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 300 agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactctag aggatccccg 360 ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 420 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 480 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct 540 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 600 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 660 tgccgcgttt ataatgaaga ccctagcagg catcaaataa aacgaaaggc tcagtcgaaa 720 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat 780 ccgccgccct agacagctgt cgctgagacg atcgctgaga cctcgcaagt tctcgccatc 840 gcaggtgaaa tctagatgta ttcgc 865 <210> 74 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR Amplification Promoter PaprE <400> 74 tatatgaaga ccttcgactc gggacctctt tccctcg 37 <210> 75 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR Amplification Promoter PaprE <400> 75 tatagaagac ttatatctca ctctcctcct ctttattcag a 41 <210> 76 <211> 747 <212> DNA <213> Artificial Sequence <220> <223> Functional fragment: GFPmut2 AF302837 flanked by BpiI restriction sites <400> 76 attagaagac ctatatgagt aaaggagaag aacttttcac tggagttgtc ccaattcttg 60 ttgaattaga tggcgatgtt aatgggcaaa aattctctgt cagtggagag ggtgaaggtg 120 atgcaacata cggaaaactt acccttaaat ttatttgcac tactgggaag ctacctgttc 180 catggccaac acttgtcact actttcgcgt atggtcttca atgctttgcg agatacccag 240 atcatatgaa acagcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa 300 gaactatatt ttacaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag 360 gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca 420 ttcttggaca caaaatggaa tacaactata actcacataa tgtatacatc atggcagaca 480 aaccaaagaa tggaatcaaa gttaacttca aaattagaca caacattaaa gatggaagcg 540 ttcaattagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac 600 cagacaacca ttacctgtcc acacaatctg ccctttccaa agatcccaac gaaaagagag 660 atcacatgat ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac 720 tatacaaata atagcttgtc ttcatta 747 <210> 77 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of amyB genomic region <400> 77 tataggtctc aacccataat gccgtcgcac tgg 33 <210> 78 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of amyB genomic region <400> 78 ccttggtctc gtcgctagaa gagcagagag gacgg 35 <210> 79 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of amyB genomic region <400> 79 ttgagaggtc tcagagacat tttccctata ttttcttcc 39 <210> 80 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of amyB genomic region <400> 80 gtgaggtctc atgagaccat ccgttattga caagg 35 <210> 81 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Protospacer: Protospacer target sequence for the sigE gene <400> 81 cggtgaggaa aaaacccaaa 20 <210> 82 <211> 1338 <212> DNA <213> Artificial sequence <220> <223> sgRNA and homology region: Protospacer epr-sgRNA, Homology region of the sigE gene with flanking BsaI sites <400> 82 ggttgcggtc tcatacgcgg tgaggaaaaa acccaaagtt ttagagctag aaatagcaag 60 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180 ggcccagtcg atacgtaaag actgggcctt tttaatacga ctcactatag ggtcgacggc 240 caacgaggcc tcgaggatcc gatatcatgc atggcgcgcc accctgagac gaccctgatg 300 caggtgaccc ggatccacta gtaagacagt atgatgaaca agtgcttgtg gaactacaca 360 ttcacggaga gacaattcgt ttaaagggac tcgtcgattc tggcaaccag ctgtatgatc 420 ctatgaccaa aacaccggtc atgatcgtcc aggccgacca tctaacggcc atttgcggag 480 aatcgtttat agaccttatg aaacagtctc atcctgttga agtcatgcaa aagatcgatg 540 atcaatttcc tcttcttgat cgattaagac ttgttccata tcgagcagtc ggtcatgatc 600 acggttttct actatgccta aaaccagata cagttgtcat ttattcaaag acgcatatga 660 ttcagccagc taagtgtttt gtaggattga gtctgagcgg cttatcggca gatcaggaat 720 ttcaatccat cattcatcca gatatgttag acgggaaaat catccagggg gtgtcgtagt 780 ttttggtgat gtcttttatc ttacgaggtc aacttgacat tttgaaaaat ttttttgaaa 840 agctctgtcc ctgtactgtc aaaggaaaca accttttttc tcatgattct cgtcatcgct 900 cgtgcatatt tttccaaccc aaggagatac tgaactttgt acaacagctc ctgtagggag 960 ggaaaaaagt gtccagaaat aaagtggaaa tctgcggagt cgacacctcc aagctgcctg 1020 ttctgaaaaa cgacgagatg agaaaattgt tcagacagct gcaagatgag ggtgacgata 1080 cagcaagaga aaagctagtc aatggcaatt tacggttggt tttaagtgtg attcagcgtt 1140 tcaacaacag aggcgagtat gtcgatgatc tctttcaagt aggctgtatc ggattaatga 1200 aatcaattga taattttgat ttaagccaca atgtcagatt ttcaacttat gcggttccta 1260 tgatcatagg agaaatccgt cgatacttgc gtgataataa tccgattcgg gtgtctcgct 1320 cactcaagag acccgttg 1338 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Protospacer: Protospacer target sequence for the sigF gene <400> 83 tgtcgtcctc gctcaagaag 20 <210> 84 <211> 1338 <212> DNA <213> Artificial Sequence <220> <223> sgRNA and homology region: Protospacer epr-sgRNA, Homology region of the sigF gene with flanking BsaI sites <400> 84 ggttgcggtc tcatacgtgt cgtcctcgct caagaaggtt ttagagctag aaatagcaag 60 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180 ggcccagtcg atacgtaaag actgggcctt tttaatacga ctcactatag ggtcgacggc 240 caacgaggcc tcgaggatcc gatatcatgc atggcgcgcc accctgagac gaccctgatg 300 caggtgaccc ggatccacta gtaaattatc cgtatggagc cttcagagca aacggcattg 360 caaacattgg gggtggcatc atgaggaatg aaatgaacct gaccttctct gccttaagtc 420 aaaatgaatc ctttgcgagg gtgacagtcg cggcgtttat cgcccagctt gatccgacat 480 tagatgaatt aactgaaatc aaaaccgttg tgtcagaggc ggtgacaaac tccattatcc 540 atggctatga tgggaatcca gatggcaagg tgcatattga agtcacactt gatgatcatg 600 ttgtgtacct gaccatccgt gacgaaggaa tgggtattac agatcttgag gaagcaagac 660 agccgctttt cacgacaaaa ccagacttag aacgctctgg catgggcttt accattatgg 720 agaattttat ggatgatgtc atgatagact catctccaga aatgggcaca accatccgtt 780 taacaaagca tctatcaaaa agcaaagcgc tttgtaatta aatagccaat tcggctggct 840 ttttttgtgt ggtaattacc ggtaaatgaa gttctctcgg tatgagaacc atttttcacc 900 acatactatt tttaacccat cgtataggaa gtgacttgga tggacggaca aatctttctt 960 cggctgcgcc atcgcattaa gaccggtaat gatcagctca tttatttaga agatatcgcc 1020 caaatcactg gtgatgagtt ggctgtgcaa aagcttagca agatgccgat atatcatgtc 1080 agtaaaaagg atcgtcacat tgccgttctt gatatcatgc atgtggtcaa aacgatcaaa 1140 aaaacatggc caaccatcga cattcaaact gtcggaggcg ctgaagccat tgttgaaatt 1200 gatacaggca aacgccagct ttctcccgta ttatttgtgt tcgtgtggct tttattattt 1260 gtcggagcgg cgcttgccat tatgaatttc cacgaggatg tcagtatgcg gctcgtccat 1320 atctcaagag acccgttg 1338 <210> 85 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Protospacer: protospacer target sequence for spoIIE gene <400> 85 cttgtagctg aacagctgat 20 <210> 86 <211> 1338 <212> DNA <213> Artificial sequence <220> <223> sgRNA and homology region: protospacer epr-sgRNA, spoIIE gene homology region with flanking BsaI sites <400> 86 ggttgcggtc tcatacgctt gtagctgaac agctgatgtt ttagagctag aaatagcaag 60 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgctttttac 120 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatc taaactagta 180 ggcccagtcg atacgtaaag actgggcctt tttaatacga ctcactatag ggtcgacggc 240 caacgaggcc tcgaggatcc gatatcatgc atggcgcgcc accctgagac gaccctgatg 300 caggtgaccc ggatccacta gtcctgctgt ccgcaggtgc tttttttctt gacccacacg 360 acattttttg agatttcgtc atttaattta aaacttccta ttgacggaca agcgattcct 420 ttgtattata gatcttgtgc ttcttagcgc atttttatta tggcggtgta gctcagctgg 480 ctagagcgta cggttcatac ccgtgaggtc gggggttcga tcccctccgc cgctatcctt 540 ttgattagaa cataaaagca aggcccgttg gtcaagcggt taagacaccg ccctttcacg 600 gcggtaacac gggttcgaat cccgtacggg tcatcttcga aaacagcttt ctttaggaaa 660 gctgtttttt tgtgtcttca taaaattctg atgaaagaca tcgactttca agaaagtatg 720 cctctttgac gaataaagcg tcgaacgttt tatggaaacg acaacttctt ttgacaaaat 780 ttctttttca ccttcgctat aatgacaagc aacgaatatc agtgaaatat cgtataatat 840 gaatttcttc tggcgatgat ggggatataa agcattcagt acgatcccag gaggaatgaa 900 gatgcgaaaa ggtcacgtaa accaaatctt attgattaca gatggctgct caaatcacgg 960 ggaagatcca cttgcgattg cctcattggc aaaggaacaa gggattacag tcaatgttat 1020 tggcattatg gaggaaaaca gacacgacca tgaagcaatg aaagaagttg aagggattgc 1080 tctcgcaggt ggaggcatcc atcaagttgt ctacgtccag cagttatctc aaaccgtaca 1140 aatggttaca aaaaaagcga tgacacaaac cttgcaaggt gttgtgaata aagaattgca 1200 gcaaatactt ggcaaggaca ctgaaattga agagctgcca cctgataaac gcggggaagt 1260 gatggaagta gtcgatgagt taggagagac ggttcatctt caagtgcttg tgcttgttga 1320 tactcaagag acccgttg 1338 <210> 87 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of hag genomic region <400> 87 tgatcttgat gaaacgacgg 20 <210> 88 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of hag genomic region <400> 88 taatcctgat attctgatcg cc 22 <210> 89 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of degU genomic region <400> 89 atgatttaag gccgatggc 19 <210> 90 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of degU genomic region <400> 90 atccgccttc agctactact t 21 <210> 91 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of amyE genomic region <400> 91 ggtcatgaat aatctgcgta atagac 26 <210> 92 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR Amplification of amyE Genomic Region <400> 92 gcgtgtacgt tttgaggcgc tgcgcc 26 <210> 93 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR Amplification of aprE Genomic Region Bacillus subtilis <400> 93 gagctggcag atgaagccaa tattcc 26 <210> 94 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR Amplification of aprE Genomic Region in Bacillus subtilis <400> 94 gtacgcgcat gaggaacgac aaataag 27 <210> 95 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR Amplification of vpr Genomic Region <400> 95 ctgtcaccca cttcccatta tgag 24 <210> 96 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR Amplification of vpr Genomic Region <400> 96 gtgaccgaag gctttccatc attg 24 <210> 97 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of epr genomic region <400> 97 cttgtcatcg tcgtcgggat tcag 24 <210> 98 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of epr genomic region <400> 98 gtgccaatca caaatgtagc cagc 24 <210> 99 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of sigE genomic region <400> 99 gaggaggcat gatcggagtt cattc 25 <210> 100 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of sigE genomic region <400> 100 ctcctccgtc attatagatc ggttc 25 <210> 101 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide: PCR amplification of sigF genomic region <400> 101 gtgacgaatt atttggaaac agagg 25 <210> 102 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of sigF genomic region <400> 102 cgacataatg atcgagatcg agctg 25 <210> 103 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of spoIIE genomic region <400> 103 ctcaacaaca acaatcaaga ccgag 25 <210> 104 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Oligonucleotide: PCR amplification of spoIIE genomic region <400> 104 gatagtgaat cgaatcgagg cgtcc 25

Claims

1. A shuttle vector comprising a. a high-copy replication origin functional in Escherichia coli, and b. a low- to medium-copy replication origin functional in Bacillus, and c. a synthetic constitutive regulatory nucleic acid that confers reduced constitutive expression compared to the corresponding initiating regulatory nucleic acid molecule in a bacterial cell, wherein the initiating regulatory nucleic acid molecule that confers constitutive expression in a bacterial cell is selected from i. SEQ ID NO: 28 and 29 or functional variants thereof, wherein the functional variants are the sequences shown in SEQ ID NO 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47; and ii. the complementary sequence of any one of the nucleic acid molecules as defined in i); wherein the synthetic constitutive regulatory nucleic acid is operably linked to a coding region that, upon high expression, will stress the bacterium, thereby resulting in a reduced growth rate or growth potential of the bacterium, wherein the low- to medium-copy replication origin functional in Bacillus is a temperature-sensitive derivative of the pE194 replication origin.

2. The shuttle vector according to claim 1, wherein the temperature-sensitive derivative of the pE194 replication origin is the pE194ts replication origin.

3. The shuttle vector according to claim 1, wherein the coding region encodes a CRISPR / Cas enzyme.

4. The shuttle vector according to claim 3, wherein the CRISPR / Cas enzyme is a Cas9 or Cas12a enzyme.

5. The shuttle vector according to claim 1, wherein the synthetic constitutive regulatory nucleic acid confers expression in Bacillus.

6. The shuttle vector according to any one of claims 1 to 5, wherein the synthetic constitutive regulatory nucleic acid is selected from the group consisting of a. nucleic acid molecules of the sequences shown in SEQ ID NO 35, 36, 37, 38, 39, 40, 42, 43, 45, 46 or 47, and b. the complementary sequence of any one of the nucleic acid molecules as defined in a), wherein the sequence as defined in b) is different from the corresponding initiating nucleic acid molecule.

7. A method for expressing a coding region in a bacterium, wherein, upon high expression, the coding region will stress the bacterium, thereby resulting in a reduced growth rate or growth potential of the bacterium, the method comprising introducing into the bacterium a shuttle vector according to any one of claims 1 to 6, wherein the coding region is operably linked to the synthetic constitutive regulatory nucleic acid that confers reduced constitutive expression, and wherein the bacterium is Escherichia coli, Bacillus subtilis, Bacillus licheniformis or Bacillus pumilus.

8. The method according to claim 7, wherein the coding region is a protein essential for genome editing, and wherein the coding region encodes a CRISPR / Cas enzyme.

9. The method according to claim 8, wherein the CRISPR / Cas enzyme is a Cas9 or Cas12a enzyme.

10. The method according to claim 7, wherein the bacterium is Bacillus licheniformis.

11. A system for expressing a coding region encoding a protein, the expression of which will stress the bacterium, the system comprising the shuttle vector according to any one of claims 1 to 6 and a coding region heterologous to the constitutive regulatory nucleic acid, which confers reduced constitutive expression compared to the corresponding starting regulatory nucleic acid molecule in the bacterial cell, wherein the coding region encodes a protein essential for genome editing and is a CRISPR / Cas enzyme.

12. The system according to claim 11, wherein the CRISPR / Cas enzyme is a Cas9 or Cas12a enzyme.

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